Flow cell with selective deposition or activation of nucleotides

ABSTRACT

An apparatus includes a flow cell body, a plurality of electrodes, an integrated circuit, and an imaging assembly. The flow cell body defines one or more flow channels and a plurality of wells. Each flow channel is configured to receive a flow of fluid. Each well is fluidically coupled with the corresponding flow channel. Each well is configured to contain at least one polynucleotide. Each electrode is positioned in a corresponding well of the plurality of wells. The electrodes are operable to effect writing of polynucleotides in the corresponding wells. The integrated circuit is operable to drive selective deposition or activation of selected nucleotides to attach to polynucleotides in the wells to thereby generate polynucleotides representing machine-written data in the wells. The imaging assembly is operable to capture images indicative of one or more nucleotides in a polynucleotide.

RELATED APPLICATION

This application claims priority to U.S. Provisional Patent App. No.62/855,657, entitled “Flow Cell with Selective Deposition or Activationof Nucleotides,” filed on May 31, 2019, which is incorporated byreference herein in its entirety.

BACKGROUND

Computer systems have used various different mechanisms to store data,including magnetic storage, optical storage, and solid-state storage.Such forms of data storage may present drawbacks in the form ofread-write speed, duration of data retention, power usage, or datadensity.

Just as naturally occurring DNA may be read, machine-written DNA mayalso be read. Pre-existing DNA reading techniques may include anarray-based, cyclic sequencing assay (e.g., sequencing-by-synthesis(SBS)), where a dense array of DNA features (e.g., template nucleicacids) are sequenced through iterative cycles of enzymatic manipulation.After each cycle, an image may be captured and subsequently analyzedwith other images to determine a sequence of the machine-written DNAfeatures. In another biochemical assay, an unknown analyte having anidentifiable label (e.g., fluorescent label) may be exposed to an arrayof known probes that have predetermined addresses within the array.Observing chemical reactions that occur between the probes and theunknown analyte may help identify or reveal properties of the analyte.

SUMMARY

Described herein are devices, systems, and methods for selectivelyactivating or depositing nucleotides within a flow cell.

An implementation relates to an apparatus comprising a flow cell bodydefining one or more flow channels and a plurality of wells, each flowchannel of the one or more flow channels to receive a flow of fluid. Insome such implementations, each well of the plurality of wells may befluidically coupled with the corresponding flow channel of the one ormore flow channels, each well of the plurality of wells to contain atleast one polynucleotide. In some such implementations, the apparatusmay comprise a plurality of electrodes. In some such implementations,each electrode of the plurality of electrodes may be positioned in acorresponding well of the plurality of wells, the plurality ofelectrodes to effect writing of polynucleotides in the correspondingwells of the plurality of wells. In some such implementations, theapparatus may comprise an integrated circuit, the integrated circuit todrive selective deposition or activation of selected nucleotides toattach to polynucleotides in the wells of the plurality of wells tothereby generate polynucleotides representing machine-written data inthe plurality of wells. In some such implementations, the apparatus maycomprise an imaging assembly to capture images indicative of one or morenucleotides in a polynucleotide.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may define anaperture.

Variations on any one or more of the above implementations exist,wherein the imaging assembly may include at least one image sensor toreceive light through the aperture of each electrode of the plurality ofelectrodes.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may be annularlyshaped.

Variations on any one or more of the above implementations exist,wherein the plurality of electrodes may comprise a plurality ofelectrode segments arranged in quadrants, the aperture being defined ata central region of the arrangement of quadrants.

Variations on any one or more of the above implementations exist,wherein the integrated circuit may be further in communication with theimaging assembly.

Variations on any one or more of the above implementations exist,wherein the integrated circuit may comprise a CMOS chip.

Variations on any one or more of the above implementations exist,wherein the plurality of wells may be formed as a plurality of discreterecesses arranged in a pattern along a floor of the corresponding flowchannel of the one or more flow channels.

Variations on any one or more of the above implementations exist,wherein each well of the plurality of wells may be defined by at leastone sidewall and a floor.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may be positionedon the floor of the corresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each electrode of the plurality of electrodes may be positionedon a sidewall of the at least one sidewall of the corresponding well.

Variations on any one or more of the above implementations exist,wherein the floor of each well of the plurality of wells may furtherdefine an aperture, the aperture to provide a path for fluidcommunication between the well of the plurality of wells and a fluidsource.

Variations on any one or more of the above implementations exist,wherein the integrated circuit to drive the plurality of electrodes toselectively deposit or activate selected nucleotides by applying avoltage within the corresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein each nucleotide may be associated with a particular voltage, theintegrated circuit to drive the electrodes of the plurality ofelectrodes to selectively deposit or activate a selected nucleotide byapplying the particular voltage associated with the selected nucleotide.

Variations on any one or more of the above implementations exist,wherein each well of the plurality of wells may include a set of fourelectrodes from the plurality of electrodes, each electrode in the setof four being associated with a corresponding voltage of the particularvoltages associated with the nucleotides, such that each electrode inthe set of four corresponds with a particular one of four nucleotides.

Variations on any one or more of the above implementations exist,wherein the integrated circuit to drive the selective deposition oractivation of selected nucleotides by applying a change in pH within thecorresponding well of the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise at least one light source,the integrated circuit to drive the selective deposition or activationof selected nucleotides by activating the at least one light source.

Variations on any one or more of the above implementations exist,wherein each nucleotide may be associated with a particular wavelengthof light, the integrated circuit to drive the at least one light sourceto selectively deposit or activate a selected nucleotide by applying theparticular wavelength of light associated with the selected nucleotide.

Variations on any one or more of the above implementations exist,wherein the integrated circuit to drive the selective deposition oractivation of selected nucleotides by applying a change in pH within thecorresponding well of the plurality of wells in addition to driving theactivation of the at least one light source.

Variations on any one or more of the above implementations exist,wherein the at least one light source may comprise a light matrix.

Variations on any one or more of the above implementations exist,wherein the light matrix may comprise a matrix of microscopic lightemitting diodes.

Variations on any one or more of the above implementations exist,wherein the light matrix to project light onto a bottom of each well ofthe plurality of wells.

Variations on any one or more of the above implementations exist,wherein the light matrix may be positioned under a bottom of each wellof the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise one or more polarizers, theintegrated circuit to drive the selective deposition or activation ofselected nucleotides by activating the at least one polarizer of the oneor more polarizers in coordination with the at least one light source.

Variations on any one or more of the above implementations exist,wherein the integrated circuit to drive the selective deposition oractivation of selected nucleotides by activating communication ofpre-charged enzymes to the one or more flow channels.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise a printhead, the integratedcircuit to drive the selective deposition or activation of selectednucleotides by activating the printhead.

Variations on any one or more of the above implementations exist,wherein each nozzle of the four nozzles to dispense a correspondingnucleotide.

Variations on any one or more of the above implementations exist,wherein the integrated circuit further to drive acoustic tamping ofdroplets emitted by the printhead.

Variations on any one or more of the above implementations exist,wherein the integrated circuit further to activate the printhead and theplurality of electrodes in cooperation to thereby drive the selectivedeposition or activation of selected nucleotides.

Another implementation relates to an apparatus comprising a flow cellbody defining one or more flow channels and a plurality of wells, eachflow channel of the one or more flow channels to receive a flow offluid. In some such implementations, each well of the plurality of wellsmay be fluidically coupled with the corresponding flow channel of theone or more flow channels, each well of the plurality of wells tocontain at least one polynucleotide. In some such implementations, theapparatus may comprise a plurality of electrodes. In some suchimplementations, each electrode of the plurality of electrodes may bepositioned in a corresponding well of the plurality of wells, theplurality of electrodes to effect writing of polynucleotides in thecorresponding wells of the plurality of wells. In some suchimplementations, the apparatus may comprise an integrated circuit, theintegrated circuit to drive selective deposition or activation ofselected nucleotides to attach to polynucleotides in the plurality ofwells to thereby generate polynucleotides representing machine-writtendata in the plurality of wells, each nucleotide being associated with aparticular voltage, the integrated circuit to drive the plurality ofelectrodes to selectively deposit or activate a selected nucleotide byapplying the particular voltage associated with the selected nucleotide.

Variations on any one or more of the above implementations exist,wherein each well of the plurality of wells may include a set of fourelectrodes from the plurality of electrodes, each electrode in the setof four being associated with a corresponding voltage of the particularvoltages associated with the nucleotides, such that each electrode inthe set of four corresponds with a particular one of four nucleotides.

Yet another implementation relates to an apparatus comprising a flowcell body defining one or more flow channels and a plurality of wells,each flow channel of the one or more flow channels to receive a flow offluid. In some such implementations, each well of the plurality of wellsmay be fluidically coupled with the corresponding flow channel of theone or more flow channels, each well of the plurality of wells tocontain at least one polynucleotide. In some such implementations, theapparatus may comprise a plurality of electrodes. In some suchimplementations, each electrode of the plurality of electrodes may bepositioned in a corresponding well of the plurality of wells, theplurality of electrodes to effect writing of polynucleotides in thecorresponding wells of the plurality of wells. In some suchimplementations, the apparatus may comprise at least one light source.In some such implementations, the apparatus may comprise an integratedcircuit, the integrated circuit to drive selective deposition oractivation of selected nucleotides to attach to polynucleotides in theplurality of wells to thereby generate polynucleotides representingmachine-written data in the plurality of wells, the integrated circuitto drive the selective deposition or activation of selected nucleotidesby activating the at least one light source.

Variations on any one or more of the above implementations exist,wherein each nucleotide may be associated with a particular wavelengthof light, the integrated circuit to drive the at least one light sourceto selectively deposit or activate a selected nucleotide by applying theparticular wavelength of light associated with the selected nucleotide.

Variations on any one or more of the above implementations exist,wherein the integrated circuit to drive the selective deposition oractivation of selected nucleotides by applying a change in pH within thecorresponding well of the plurality of wells in addition to driving theactivation of the at least one light source.

Variations on any one or more of the above implementations exist,wherein the at least one light source may comprise a light matrix.

Variations on any one or more of the above implementations exist,wherein the light matrix may comprise a matrix of micro-LEDs.

Variations on any one or more of the above implementations exist,wherein the light matrix to project light onto a bottom of each well ofthe plurality of wells.

Variations on any one or more of the above implementations exist,wherein the light matrix may be positioned under a bottom of each wellof the plurality of wells.

Variations on any one or more of the above implementations exist,wherein the apparatus may further comprise one or more polarizers, theintegrated circuit to drive the selective deposition or activation ofselected nucleotides by activating the one or more polarizers incoordination with the at least one light source.

Yet another implementation relates to an apparatus comprising a flowcell body defining one or more flow channels and a plurality of wells,each flow channel of the one or more flow channels to receive a flow offluid. In some such implementations, each well of the plurality of wellsmay be fluidically coupled with the corresponding flow channel of theone or more flow channels, each well of the plurality of wells tocontain at least one polynucleotide. In some such implementations, theapparatus may comprise a plurality of electrodes. In some suchimplementations, each electrode of the plurality of electrodes may bepositioned in a corresponding well of the plurality of wells, theplurality of electrodes to effect writing of polynucleotides in thecorresponding wells of the plurality of wells. In some suchimplementations, the apparatus may comprise a printhead to depositnucleotides. In some such implementations, the apparatus may comprise anintegrated circuit, the integrated circuit to drive selective depositionof selected nucleotides to attach to polynucleotides in the plurality ofwells to thereby generate polynucleotides representing machine-writtendata in the plurality of wells, the integrated circuit to drive theselective deposition of selected nucleotides by activating theprinthead.

Variations on any one or more of the above implementations exist,wherein the printhead may include four nozzles, each nozzle to dispensea corresponding nucleotide.

Variations on any one or more of the above implementations exist,wherein the integrated circuit further to drive acoustic tamping ofdroplets emitted by the printhead.

Variations on any one or more of the above implementations exist,wherein the integrated circuit further to activate the printhead and theplurality of electrodes in cooperation to thereby drive the selectivedeposition or activation of selected nucleotides.

Yet another implementation relates to a method comprising flowing afluid through a flow channel of a flow cell. In some suchimplementations, the fluid may comprise a plurality of types ofnucleotides, the flow cell may include a plurality of primary bases tosupport polynucleotides, the primary bases being secured to a floorregion of the flow cell. In some such implementations, the method maycomprise selecting one type of nucleotide for attachment to a selectedprimary base of the plurality of primary bases in the flow cell. In somesuch implementations, the method may comprise activating the selectedone type of nucleotide to thereby cause the selected one type ofnucleotide to attach to the selected primary base, the attachednucleotide representing machine-written data.

Variations on any one or more of the above implementations exist,wherein the method may further comprise repeating selecting one type ofnucleotide for attachment to a selected primary base in the flow celland activating the selected one type of nucleotide to thereby cause theselected one type of nucleotide to attach to the selected primary base,to thereby generate a polynucleotide on the primary base, the generatedpolynucleotide including a plurality of selected types of nucleotides,the generated polynucleotide representing machine-written data.

Variations on any one or more of the above implementations exist,wherein activating the selected type of nucleotide may compriseactivating an electrode in the flow cell to thereby apply a voltage tothe selected type of nucleotide.

Variations on any one or more of the above implementations exist,wherein, each type of nucleotide may be associated with a particularvoltage, and activating the electrode may comprise activating theelectrode to apply the particular voltage associated with the selectedtype of nucleotide.

I Variations on any one or more of the above implementations exist,wherein the flow cell may include electrodes associated with differentcorresponding voltages, and the method may further comprise selecting anelectrode from the electrodes, the selected electrode corresponding tothe voltage associated with the selected type of nucleotide, theactivated electrode being the selected electrode.

Variations on any one or more of the above implementations exist,wherein activating the selected type of nucleotide may comprise applyinga change in pH within the flow cell, the applied pH being associatedwith the selected type of nucleotide.

Variations on any one or more of the above implementations exist,wherein activating the selected type of nucleotide may compriseactivating at least one light source.

Variations on any one or more of the above implementations exist,wherein each type of nucleotide may be associated with a particularwavelength of light, and activating at least one light source maycomprise activating the at least one light source to emit light at theparticular wavelength associated with the selected type of nucleotide.

Variations on any one or more of the above implementations exist,wherein activating the selected type of nucleotide may further compriseapplying a change in pH within the flow cell in coordination withactivating the at least one light source, the applied pH beingassociated with the selected type of nucleotide.

Variations on any one or more of the above implementations exist,wherein activating the selected type of nucleotide may further compriseactivating at least one polarizer in coordination with activating the atleast one light source.

Variations on any one or more of the above implementations exist,wherein activating the selected type of nucleotide may comprisecommunicating pre-charged enzymes to the flow channel.

Yet another implementation relates to a method comprising selecting onetype of nucleotide for attachment to a selected primary base in a flowcell. In some such implementations, the method may comprise depositingthe selected one type of nucleotide into the flow cell, the flow cellincluding a plurality of primary bases to support polynucleotides, theprimary bases being secured to a floor region of the flow cell, thedeposited nucleotide attaching to a corresponding primary base of theplurality of primary bases, the attached nucleotide representingmachine-written data.

Variations on any one or more of the above implementations exist,wherein, depositing the selected type of nucleotide may compriseemitting the selected type of nucleotide from a printhead.

Variations on any one or more of the above implementations exist,wherein the printhead may include a plurality of nozzles, each nozzle ofthe plurality of nozzles being associated with a particular type ofnucleotide. In some such implementations, the method may furthercomprise selecting the nozzle of the plurality of nozzles correspondingto the selected type of nucleotide, the deposited nucleotide beingdeposited from the selected nozzle of the plurality of nozzles.

Variations on any one or more of the above implementations exist,wherein depositing the selected type of nucleotide may further compriseactivating an electrode in the flow cell in coordination with emittingthe selected type of nucleotide from the printhead.

Variations on any one or more of the above implementations exist,wherein the flow cell may include electrodes associated with differentcorresponding voltages, each type of nucleotide being associated with aparticular voltage. In some such implementations, the method may furthercomprise selecting an electrode of the electrodes, the selectedelectrode corresponding to the voltage associated with the selected typeof nucleotide, the activated electrode being the selected electrode.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein and to achieve thebenefits/advantages as described herein. In particular, all combinationsof claimed subject matter appearing at the end of this disclosure arecontemplated as being part of the inventive subject matter disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of one or more implementations are set forth in theaccompanying drawings and the description below. Other features,aspects, and advantages will become apparent from the description, thedrawings, and the claims, in which:

FIG. 1 depicts a block schematic view of an example of a system that maybe used to conduct biochemical processes;

FIG. 2 depicts a block schematic cross-sectional view of an example of aconsumable cartridge that may be utilized with the system of FIG. 1;

FIG. 3 depicts a perspective view of an example of a flow cell that maybe utilized with the system of FIG. 1;

FIG. 4 depicts an enlarged perspective view of a channel of the flowcell of FIG. 3;

FIG. 5 depicts a block schematic cross-sectional view of an example ofwells that may be incorporated into the channel of FIG. 4;

FIG. 6 depicts a flow chart of an example of a process for readingpolynucleotides;

FIG. 7 depicts a block schematic cross-sectional view of another exampleof wells that may be incorporated into the channel of FIG. 4;

FIG. 8 depicts a flow chart of an example of a process for writingpolynucleotides;

FIG. 9 depicts a top plan view of an example of an electrode assembly;

FIG. 10 depicts a block schematic cross-sectional view of anotherexample of wells that may be incorporated into the channel of FIG. 4;

FIG. 11 depicts a block schematic cross-sectional view of anotherexample of a flow cell that may be utilized with the system of FIG. 1;

FIG. 12 depicts a block schematic cross-sectional view of anotherexample of a flow cell that may be utilized with the system of FIG. 1;and

FIG. 13 depicts a block schematic cross-sectional view of anotherexample of a flow cell that may be utilized with the system of FIG. 1.

It will be recognized that some or all of the figures are schematicrepresentations for purposes of illustration. The figures are providedfor the purpose of illustrating one or more implementations with theexplicit understanding that they will not be used to limit the scope orthe meaning of the claims.

DETAILED DESCRIPTION

In some aspects, methods and systems are disclosed herein forsynthesizing DNA (or other biological material) to store data or otherinformation by selectively depositing or activating nucleotides during awriting process in a DNA storage device. Machine-written DNA may providean alternative to traditional forms of data storage (e.g., magneticstorage, optical storage, and solid-state storage). Machine-written DNAmay provide faster read-write speeds, longer data retention, reducedpower usage, and higher data density.

Examples of how digital information may be stored in DNA are disclosedin U.S. Pub. No. 2015/0261664, entitled “High-Capacity of Storage ofDigital Information in DNA,” published Sep. 17, 2015, which isincorporated by reference herein in its entirety. For example, methodsfrom code theory to enhance the recoverability of the encoded messagesfrom the DNA segment, including forbidding DNA homopolymers (i.e. runsof more than one identical base) that are known to be associated withhigher error rates in existing high throughput technologies may be used.Further, an error-detecting component, analogous to a parity-check bit,may be integrated into the indexing information in the code. Morecomplex schemes, including but not limited to error-correcting codesand, indeed, substantially any form of digital data security (e.g.,RAID-based schemes) currently employed in informatics, may beimplemented in future developments of the DNA storage scheme. The DNAencoding of information may be computed using software. The bytescomprising each computer file may be represented by a DNA sequence withno homopolymers by an encoding scheme to produce an encoded file thatreplaces each byte by five or six bases forming the DNA sequence.

The code used in the encoding scheme may be constructed to permit astraightforward encoding that is close to the optimum informationcapacity for a run length-limited channel (e.g., no repeatednucleotides), though other encoding schemes may be used. The resultingin silico DNA sequences may be too long to be readily produced bystandard oligonucleotide synthesis and may be split into overlappingsegments of a length of 100 bases with an overlap of 75 bases. To reducethe risk of systematic synthesis errors introduced to any particular runof bases, alternate ones of the segments may be converted to theirreverse complement, meaning that each base may be “written” four times,twice in each direction. Each segment may then be augmented with anindexing information that permits determination of the computer filefrom which the segment originated and its location within that computerfile, plus simple error-detection information. This indexing informationmay also be encoded in as non-repeating DNA nucleotides and appended tothe information storage bases of the DNA segments. The division of theDNA segments into lengths of 100 bases with an overlap of 75 bases ispurely arbitrary and illustrative, and it is understood that otherlengths and overlaps may be used and is not limiting.

Other encoding schemes for the DNA segments may be used, for example toprovide enhanced error-correcting properties. The amount of indexinginformation may be increased in order to allow more or larger files tobe encoded. One extension to the coding scheme in order to avoidsystematic patterns in the DNA segments may be to add change theinformation. One way may use the “shuffling” of information in the DNAsegments, where the information may be retrieved if one knows thepattern of shuffling. Different patterns of shuffles may be used fordifferent ones of the DNA segments. A further way is to add a degree ofrandomness into the information in each one of the DNA segments. Aseries of random digits may be used for this, using modular addition ofthe series of random digits and the digits comprising the informationencoded in the DNA segments. The information may be retrieved by modularsubtraction during decoding if one knows the series of random digitsused. Different series of random digits may be used for different onesof the DNA segments The data-encoding component of each string maycontain Shannon information at 5.07 bits per DNA base, which is close tothe theoretical optimum of 5.05 bits per DNA base for base-4 channelswith run length limited to one. The indexing implementation may permit314=4782969 unique data locations. Increasing the number of indexingtrits (and therefore bases) used to specify file and intra-file locationby just two, to 16, gives 316=43046721 unique locations, in excess ofthe 16.8M that is the practical maximum for the Nested Primer MolecularMemory (NPMM) scheme.

The DNA segment designs may be synthesized in three distinct runs (withthe DNA segments randomly assigned to runs) to create approx. 1.2×10⁷copies of each DNA segment design. Phosphoramidite chemistry may beused, and inkjet printing and flow cell reactor technologies in anin-situ microarray synthesis platform may be employed. The inkjetprinting within an anhydrous chamber may allow the delivery of verysmall volumes of phosphoramidites to a confined coupling area on a 2Dplanar surface, resulting in the addition of hundreds of thousands ofbases in parallel. Subsequent oxidation and detritylation may be carriedout in a flow cell reactor. Once DNA synthesis is completed, theoligonucleotides may then be cleaved from the surface and deprotected.

Adapters may then be added to the DNA segments to enable a plurality ofcopies of the DNA segments to be made. A DNA segment with no adapter mayrequire additional chemical processes to “kick start” the chemistry forthe synthesis of the multiple copies by adding additional groups ontothe ends of the DNA segments. Oligonucleotides may be amplified usingpolymerase chain reaction (PCR) methods and paired-end PCR primers,followed by bead purification and quantification. Oligonucleotides maythen be sequenced to produce reads of 104 bases. The digital informationdecoding may then be carried out via sequencing of the central bases ofeach oligo from both ends and rapid computation of full-length oligosand removal of sequence reads inconsistent with the designs. Sequencereads may be decoded using computer software that exactly reverses theencoding process. Sequence reads for which the parity-check tritindicates an error or that may be unambiguously decoded or assigned to areconstructed computer file may be discarded. Locations within everydecoded file may be detected in multiple different sequenced DNA oligos,and simple majority voting may be used to resolve any discrepanciescaused by the DNA synthesis or the sequencing errors.

While several examples herein are provided in the context ofmachine-written DNA, it is contemplated that the principles describedherein may be applied to other kinds of machine-written biologicalmaterial.

As used herein, the term “machine-written DNA” shall be read to includeone or more strands of polynucleotides that are generated by a machine,or otherwise modified by a machine, to store data or other information.One example of the polynucleotide herein is a DNA. It is noted thatwhile the term “DNA” in the context of DNA being read or written is usedthroughout this disclosure, the term is used only as a representativeexample of a polynucleotide and may encompass the concept of apolynucleotide. “Machine,” as used herein in reference to“machine-written,” may include an instrument or system speciallydesigned for writing DNA as described in greater detail herein. Thesystem may be non-biological or biological. In one example, thebiological system may comprise, or is, a polymerase. For example, thepolymerase may be terminal deoxynucleotidyl transferase (TdT). In abiological system, the process may be additionally controlled by amachine hardware (e.g., processor) or an algorithm. “Machine-writtenDNA” may include any polynucleotide having one or more base sequenceswritten by a machine. While machine-written DNA is used herein as anexample, other polynucleotide strands may be substituted formachine-written DNA described herein. “Machine-written DNA” may includenatural bases and modifications of natural bases, including but notlimited to bases modified with methylation or other chemical tags; anartificially synthesized polymer that is similar to DNA, such as peptidenucleic acid (PNA); or Morpholino DNA. “Machine-written DNA” may alsoinclude DNA strands or other polynucleotides that are formed by at leastone strand of bases originating from nature (e.g., extracted from anaturally occurring organism), with a machine-written strand of basessecured thereto either in a parallel fashion or in an end-to-endfashion. In other implementations, “machine-written DNA” may be writtenby a biological system (e.g., enzyme) in lieu of, or in addition to, anon-biological system (e.g., the electrode machine) writing of DNAdescribed herein. In other words, “machine-written DNA” may be writtendirectly by a machine; or by an enzyme (e.g., polymerase) that iscontrolled by an algorithm and/or machine.

“Machine-written DNA” may include data that have been converted from araw form (e.g., a photograph, a text document, etc.) into a binary codesequence using known techniques, with that binary code sequence thenbeing converted to a DNA base sequence using known techniques, and withthat DNA base sequence then being generated by a machine in the form ofone or more DNA strands or other polynucleotides. Alternatively,“machine-written DNA” may be generated to index or otherwise trackpre-existing DNA, to store data or information from any other source andfor any suitable purpose, without necessarily requiring an intermediatestep of converting raw data to a binary code.

As described in greater detail below, machine-written DNA may be writtento and/or read from a reaction site. As used herein, the term “reactionsite” is a localized region where at least one designated reaction mayoccur. A reaction site may include support surfaces of a reactionstructure or substrate where a substance may be immobilized thereon. Forinstance, the reaction site may be a discrete region of space where adiscrete group of DNA strands or other polynucleotides are written. Thereaction site may permit chemical reactions that are isolated fromreactions that are in adjacent reaction sites. Devices that providemachine-writing of DNA may include flow cells with wells having writingfeatures (e.g., electrodes) and/or reading features. In some instances,the reaction site may include a surface of a reaction structure (whichmay be positioned in a channel of a flow cell) that already has areaction component thereon, such as a colony of polynucleotides thereon.In some flow cells, the polynucleotides in the colony have the samesequence, being for example, clonal copies of a single stranded ordouble stranded template. However, in some flow cells a reaction sitemay contain only a single polynucleotide molecule, for example, in asingle stranded or double stranded form.

A plurality of reaction sites may be randomly distributed along thereaction structure of the flow cells or may be arranged in apredetermined manner (e.g., side-by-side in a matrix, such as inmicroarrays). A reaction site may also include a reaction chamber,recess, or well that at least partially defines a spatial region orvolume configured to compartmentalize the designated reaction. As usedherein, the term “reaction chamber” or “reaction recess” includes adefined spatial region of the support structure (which is oftenfluidically coupled with a flow channel). A reaction recess may be atleast partially separated from the surrounding environment or otherspatial regions. For example, a plurality of reaction recesses may beseparated from each other by shared walls. As a more specific example,the reaction recesses may be nanowells comprising an indent, pit, well,groove, cavity or depression defined by interior surfaces of a detectionsurface and have an opening or aperture (i.e., be open-sided) so thatthe nanowells may be fluidically coupled with a flow channel.

A plurality of reaction sites may be randomly distributed along thereaction structure of the flow cells or may be arranged in apredetermined manner (e.g., side-by-side in a matrix, such as inmicroarrays). A reaction site may also include a reaction chamber,recess, or well that at least partially defines a spatial region orvolume configured to compartmentalize the designated reaction. As usedherein, the term “reaction chamber” or “reaction recess” includes adefined spatial region of the support structure (which is oftenfluidically coupled with a flow channel). A reaction recess may be atleast partially separated from the surrounding environment or otherspatial regions. For example, a plurality of reaction recesses may beseparated from each other by shared walls. As a more specific example,the reaction recesses may be nanowells comprising an indent, pit, well,groove, cavity or depression defined by interior surfaces of a detectionsurface and have an opening or aperture (i.e., be open-sided) so thatthe nanowells may be fluidically coupled with a flow channel.

To read the machine-written DNA, one or more discrete detectable regionsof reaction sites may be defined. Such detectable regions may beimageable regions, electrical detection regions, or other types ofregions that may have a measurable change in a property (or absence ofchange in the property) based on the type of nucleotide present duringthe reading process.

As used herein, the term “pixel” refers to a discrete imageable region.Each imageable region may include a compartment or discrete region ofspace where a polynucleotide is present. In some instances, a pixel mayinclude two or more reaction sites (e.g., two or more reaction chambers,two or more reaction recesses, two or more wells, etc.). In some otherinstances, a pixel may include just one reaction site. Each pixel isdetected using a corresponding detection device, such as an image sensoror other light detection device. The light detection device may bemanufactured using integrated circuit manufacturing processes, such asprocesses used to manufacture charged-coupled devices circuits (CCD) orcomplementary-metal-oxide semiconductor (CMOS) devices or circuits. Thelight detection device may thereby include, for example, one or moresemiconductor materials, and may take the form of, for example, a CMOSlight detection device (e.g., a CMOS image sensor) or a CCD imagesensor, another type of image sensor. A CMOS image sensor may include anarray of light sensors (e.g. photodiodes). In one implementation, asingle image sensor may be used with an objective lens to captureseveral “pixels,” during an imaging event. In some otherimplementations, each discrete photodiode or light sensor may capture acorresponding pixel. In some implementations, light sensors (e.g.,photodiodes) of one or more detection devices may be associated withcorresponding reaction sites. A light sensor that is associated with areaction site may detect light emissions from the associated reactionsite. In some implementations, the detection of light emissions may bedone via at least one light guide when a designated reaction hasoccurred at the associated reaction site. In some implementations, aplurality of light sensors (e.g., several pixels of a light detection orcamera device) may be associated with a single reaction site. In someimplementations, a single light sensor (e.g. a single pixel) may beassociated with a single reaction site or with a group of reactionsites.

As used herein, the term “synthesis” shall be read to include processeswhere DNA is generated by a machine to store data or other information.Thus, machine-written DNA may constitute synthesized DNA. As usedherein, the terms “consumable cartridge,” “reagent cartridge,”“removeable cartridge,” and/or “cartridge” refer to the same cartridgeand/or a combination of components making an assembly for a cartridge orcartridge system. The cartridges described herein may be independent ofthe element with the reaction sites, such as a flow cell having aplurality of wells. In some instances, a flow cell may be removablyinserted into a cartridge, which is then inserted into an instrument. Insome other implementations, the flow cell may be removably inserted intothe instrument without a cartridge. As used herein, the term“biochemical analysis” may include at least one of biological analysisor chemical analysis.

The term “based on” should be understood to mean that something isdetermined at least in part by the thing it is indicated as being “basedon.” To indicate that something must necessarily be completelydetermined by something else, it is described as being based exclusivelyon whatever it is completely determined by.

The term “non-nucleotide memory” should be understood to refer to anobject, device or combination of devices capable of storing data orinstructions in a form other than nucleotides that may be retrievedand/or processed by a device. Examples of “non-nucleotide memory”include solid state memory, magnetic memory, hard drives, optical drivesand combinations of the foregoing (e.g., magneto-optical storageelements).

The term “DNA storage device” should be understood to refer to anobject, device, or combination of devices configured to store data orinstructions in the form of sequences of polynucleotides such asmachine-written DNA. Examples of “DNA storage devices” include flowcells having addressable wells as described herein, systems comprisingmultiple such flow cells, and tubes or other containers storingnucleotide sequences that have been cleaved from the surface on whichthey were synthesized. As used herein, the term “nucleotide sequence” or“polynucleotide sequence” should be read to include a polynucleotidemolecule, as well as the underlying sequence of the molecule, dependingon context. A sequence of a polynucleotide may contain (or encode)information indicative of certain physical characteristics.

Implementations set forth herein may be used to perform designatedreactions for consumable cartridge preparation and/or biochemicalanalysis and/or synthesis of machine-written DNA.

I. System Overview

FIG. 1 is a schematic diagram of a system 100 that is configured toconduct biochemical analysis and/or synthesis. The system 100 mayinclude a base instrument 102 that is configured to receive andseparably engage a removable cartridge 200 and/or a component with oneor more reaction sites. The base instrument 102 and the removablecartridge 200 may be configured to interact with each other to transporta biological material to different locations within the system 100and/or to conduct designated reactions that include the biologicalmaterial in order to prepare the biological material for subsequentanalysis (e.g., by synthesizing the biological material), and,optionally, to detect one or more events with the biological material.In some implementations, the base instrument 102 may be configured todetect one or more events with the biological material directly on theremovable cartridge 200. The events may be indicative of a designatedreaction with the biological material. The removable cartridge 200 maybe constructed according to any of the cartridges described herein.

Although the following is with reference to the base instrument 102 andthe removable cartridge 200 as shown in FIG. 1, it is understood thatthe base instrument 102 and the removable cartridge 200 illustrate onlyone implementation of the system 100 and that other implementationsexist. For example, the base instrument 102 and the removable cartridge200 include various components and features that, collectively, executeseveral operations for preparing the biological material and/oranalyzing the biological material. Moreover, although the removablecartridge 200 described herein includes an element with the reactionsites, such as a flow cell having a plurality of wells, other cartridgesmay be independent of the element with the reaction sites and theelement with the reaction sites may be separately insertable into thebase instrument 102. That is, in some instances a flow cell may beremovably inserted into the removable cartridge 200, which is theninserted into the base instrument 102. In some other implementations,the flow cell may be removably inserted directly into the baseinstrument 102 without the removable cartridge 200. In still furtherimplementations, the flow cell may be integrated into the removablecartridge 200 that is inserted into the base instrument 102.

In the illustrated implementation, each of the base instrument 102 andthe removable cartridge 200 are capable of performing certain functions.It is understood, however, that the base instrument 102 and theremovable cartridge 200 may perform different functions and/or may sharesuch functions. For example, the base instrument 102 is shown to includea detection assembly 110 (e.g., an imaging device) that is configured todetect the designated reactions at the removable cartridge 200. Inalternative implementations, the removable cartridge 200 may include thedetection assembly and may be communicatively coupled to one or morecomponents of the base instrument 102. As another example, the baseinstrument 102 is a “dry” instrument that does not provide, receive, orexchange liquids with the removable cartridge 200. That is, as shown,the removable cartridge 200 includes a consumable reagent portion 210and a flow cell receiving portion 220. The consumable reagent portion210 may contain reagents used during biochemical analysis and/orsynthesis. The flow cell receiving portion 220 may include an opticallytransparent region or other detectible region for the detection assembly110 to perform detection of one or more events occurring within the flowcell receiving portion 220. In alternative implementations, the baseinstrument 102 may provide, for example, reagents or other liquids tothe removable cartridge 200 that are subsequently consumed (e.g., usedin designated reactions or synthesis procedures) by the removablecartridge 200.

As used herein, the biological material may include one or morebiological or chemical substances, such as nucleosides, nucleotides,nucleic acids, polynucleotides, oligonucleotides, proteins, enzymes,peptides, oligopeptides, polypeptides, antibodies, antigens, ligands,receptors, polysaccharides, carbohydrates, polyphosphates, nanopores,organelles, lipid layers, cells, tissues, organisms, and/or biologicallyactive chemical compound(s), such as analogs or mimetics of theaforementioned species. In some instances, the biological material mayinclude whole blood, lymphatic fluid, serum, plasma, sweat, tear,saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid,vaginal excretion, serous fluid, synovial fluid, pericardial fluid,peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid,bile, urine, gastric fluid, intestinal fluid, fecal samples, liquidscontaining single or multiple cells, liquids containing organelles,fluidized tissues, fluidized organisms, viruses including viralpathogens, liquids containing multi-celled organisms, biological swabsand biological washes. In some instances, the biological material mayinclude a set of synthetic sequences, including but not limited tomachine-written DNA, which may be fixed (e.g., attached in specificwells in a cartridge) or unfixed (e.g., stored in a tube).

In some implementations, the biological material may include an addedmaterial, such as water, deionized water, saline solutions, acidicsolutions, basic solutions, detergent solutions and/or pH buffers. Theadded material may also include reagents that will be used during thedesignated assay protocol to conduct the biochemical reactions. Forexample, added liquids may include material to conduct multiplepolymerase-chain-reaction (PCR) cycles with the biological material. Inother aspects, the added material may be a carrier for the biologicalmaterial such as cell culture media or other buffered and/or pH adjustedand/or isotonic carrier that may allow for or preserve the biologicalfunction of the biological material.

It should be understood, however, that the biological material that isanalyzed may be in a different form or state than the biologicalmaterial loaded into or created by the system 100. For example, abiological material loaded into the system 100 may include whole bloodor saliva or cell population that is subsequently treated (e.g., viaseparation or amplification procedures) to provide prepared nucleicacids. The prepared nucleic acids may then be analyzed (e.g., quantifiedby PCR or sequenced by SBS) by the system 100. Accordingly, when theterm “biological material” is used while describing a first operation,such as PCR, and used again while describing a subsequent secondoperation, such as sequencing, it is understood that the biologicalmaterial in the second operation may be modified with respect to thebiological material prior to or during the first operation. For example,sequencing (e.g. SBS) may be carried out on amplicon nucleic acids thatare produced from template nucleic acids that are amplified in a prioramplification (e.g. PCR). In this case the amplicons are copies of thetemplates and the amplicons are present in higher quantity compared tothe quantity of the templates.

In some implementations, the system 100 may automatically prepare asample for biochemical analysis based on a substance provided by theuser (e.g., whole blood or saliva or a population of cells). However, inother implementations, the system 100 may analyze biological materialsthat are partially or preliminarily prepared for analysis by the user.For example, the user may provide a solution including nucleic acidsthat were already isolated and/or amplified from whole blood; or mayprovide a virus sample in which the RNA or DNA sequence is partially orwholly exposed for processing.

As used herein, a “designated reaction” includes a change in at leastone of a chemical, electrical, physical, or optical property (orquality) of an analyte-of-interest. In particular implementations, thedesignated reaction is an associative binding event (e.g., incorporationof a fluorescently labeled biomolecule with the analyte-of-interest).The designated reaction may be a dissociative binding event (e.g.,release of a fluorescently labeled biomolecule from ananalyte-of-interest). The designated reaction may be a chemicaltransformation, chemical change, or chemical interaction. The designatedreaction may also be a change in electrical properties. For example, thedesignated reaction may be a change in ion concentration within asolution. Some reactions include, but are not limited to, chemicalreactions such as reduction, oxidation, addition, elimination,rearrangement, esterification, amidation, etherification, cyclization,or substitution; binding interactions in which a first chemical binds toa second chemical; dissociation reactions in which two or more chemicalsdetach from each other; fluorescence; luminescence; bioluminescence;chemiluminescence; and biological reactions, such as nucleic acidreplication, nucleic acid amplification, nucleic acid hybridization,nucleic acid ligation, phosphorylation, enzymatic catalysis, receptorbinding, or ligand binding. The designated reaction may also be additionor removal of a proton, for example, detectable as a change in pH of asurrounding solution or environment. An additional designated reactionmay be detecting the flow of ions across a membrane (e.g., natural orsynthetic bilayer membrane). For example, as ions flow through amembrane, the current is disrupted, and the disruption may be detected.Field sensing of charged tags may also be used; as may thermal sensingand other suitable analytical sensing techniques.

In particular implementations, the designated reaction includes theincorporation of a fluorescently labeled molecule to an analyte. Theanalyte may be an oligonucleotide and the fluorescently labeled moleculemay be a nucleotide. The designated reaction may be detected when anexcitation light is directed toward the oligonucleotide having thelabeled nucleotide, and the fluorophore emits a detectable fluorescentsignal. In alternative implementations, the detected fluorescence is aresult of chemiluminescence and/or bioluminescence. A designatedreaction may also increase fluorescence (or Forster) resonance energytransfer (FRET), for example, by bringing a donor fluorophore inproximity to an acceptor fluorophore, decrease FRET by separating donorand acceptor fluorophores, increase fluorescence by separating aquencher from a fluorophore or decrease fluorescence by co-locating aquencher and fluorophore.

As used herein, a “reaction component” includes any substance that maybe used to obtain a designated reaction. For example, reactioncomponents include reagents, catalysts such as enzymes, reactants forthe reaction, samples, products of the reaction, other biomolecules,salts, metal cofactors, chelating agents, and buffer solutions (e.g.,hydrogenation buffer). The reaction components may be delivered,individually in solutions or combined in one or more mixture, to variouslocations in a fluidic network. For instance, a reaction component maybe delivered to a reaction chamber where the biological material isimmobilized. The reaction components may interact directly or indirectlywith the biological material. In some implementations, the removablecartridge 200 is preloaded with one or more of the reaction componentsinvolved in carrying out a designated assay protocol. Preloading mayoccur at one location (e.g. a manufacturing facility) prior to receiptof the cartridge 200 by a user (e.g. at a customer's facility). Forexample, the one or more reaction components or reagents may bepreloaded into the consumable reagent portion 210. In someimplementations, the removable cartridge 200 may also be preloaded witha flow cell in the flow cell receiving portion 220.

In some implementations, the base instrument 102 may be configured tointeract with one removable cartridge 200 per session. After thesession, the removable cartridge 200 may be replaced with anotherremovable cartridge 200. In other implementations, the base instrument102 may be configured to interact with more than one removable cartridge200 per session. As used herein, the term “session” includes performingat least one of sample preparation and/or biochemical analysis protocol.Sample preparation may include synthesizing the biological material;and/or separating, isolating, modifying, and/or amplifying one or morecomponents of the biological material so that the prepared biologicalmaterial is suitable for analysis. In some implementations, a sessionmay include continuous activity in which a number of controlledreactions are conducted until (a) a designated number of reactions havebeen conducted, (b) a designated number of events have been detected,(c) a designated period of system time has elapsed, (d) signal-to-noisehas dropped to a designated threshold; (e) a target component has beenidentified; (f) system failure or malfunction has been detected; and/or(g) one or more of the resources for conducting the reactions hasdepleted. Alternatively, a session may include pausing system activityfor a period of time (e.g., minutes, hours, days, weeks) and latercompleting the session until at least one of (a)-(g) occurs.

An assay protocol may include a sequence of operations for conductingthe designated reactions, detecting the designated reactions, and/oranalyzing the designated reactions. Collectively, the removablecartridge 200 and the base instrument 102 may include the components forexecuting the different operations. The operations of an assay protocolmay include fluidic operations, thermal-control operations, detectionoperations, and/or mechanical operations.

A fluidic operation includes controlling the flow of fluid (e.g., liquidor gas) through the system 100, which may be actuated by the baseinstrument 102 and/or by the removable cartridge 200. In one example,the fluid is in liquid form. For example, a fluidic operation mayinclude controlling a pump to induce flow of the biological material ora reaction component into a reaction chamber.

A thermal-control operation may include controlling a temperature of adesignated portion of the system 100, such as one or more portions ofthe removable cartridge 200. By way of example, a thermal-controloperation may include raising or lowering a temperature of a polymerasechain reaction (PCR) zone where a liquid that includes the biologicalmaterial is stored.

A detection operation may include controlling activation of a detectoror monitoring activity of the detector to detect predeterminedproperties, qualities, or characteristics of the biological material. Asone example, the detection operation may include capturing images of adesignated area that includes the biological material to detectfluorescent emissions from the designated area. The detection operationmay include controlling a light source to illuminate the biologicalmaterial or controlling a detector to observe the biological material.

A mechanical operation may include controlling a movement or position ofa designated component. For example, a mechanical operation may includecontrolling a motor to move a valve-control component in the baseinstrument 102 that operably engages a movable valve in the removablecartridge 200. In some cases, a combination of different operations mayoccur concurrently. For example, the detector may capture images of thereaction chamber as the pump controls the flow of fluid through thereaction chamber. In some cases, different operations directed towarddifferent biological materials may occur concurrently. For instance, afirst biological material may be undergoing amplification (e.g., PCR)while a second biological material may be undergoing detection.

Similar or identical fluidic elements (e.g., channels, ports,reservoirs, etc.) may be labeled differently to more readily distinguishthe fluidic elements. For example, ports may be referred to as reservoirports, supply ports, network ports, feed port, etc. It is understoodthat two or more fluidic elements that are labeled differently (e.g.,reservoir channel, sample channel, flow channel, bridge channel) do notrequire that the fluidic elements be structurally different. Moreover,the claims may be amended to add such labels to more readily distinguishsuch fluidic elements in the claims.

A “liquid,” as used herein, is a substance that is relativelyincompressible and has a capacity to flow and to conform to a shape of acontainer or a channel that holds the substance. A liquid may beaqueous-based and include polar molecules exhibiting surface tensionthat holds the liquid together. A liquid may also include non-polarmolecules, such as in an oil-based or non-aqueous substance. It isunderstood that references to a liquid in the present application mayinclude a liquid comprising the combination of two or more liquids. Forexample, separate reagent solutions may be later combined to conductdesignated reactions.

One or more implementations may include retaining the biologicalmaterial (e.g., template nucleic acid) at a designated location wherethe biological material is analyzed. As used herein, the term“retained,” when used with respect to a biological material, includesattaching the biological material to a surface or confining thebiological material within a designated space. As used herein, the term“immobilized,” when used with respect to a biological material, includesattaching the biological material to a surface in or on a solid support.Immobilization may include attaching the biological material at amolecular level to the surface. For example, a biological material maybe immobilized to a surface of a substrate using adsorption techniquesincluding non-covalent interactions (e.g., electrostatic forces, van derWaals, and dehydration of hydrophobic interfaces) and covalent bindingtechniques where functional groups or linkers facilitate attaching thebiological material to the surface. Immobilizing a biological materialto a surface of a substrate may be based upon the properties of thesurface of the substrate, the liquid medium carrying the biologicalmaterial, and the properties of the biological material itself. In somecases, a substrate surface may be functionalized (e.g., chemically orphysically modified) to facilitate immobilizing the biological materialto the substrate surface. The substrate surface may be first modified tohave functional groups bound to the surface. The functional groups maythen bind to the biological material to immobilize the biologicalmaterial thereon. In some cases, a biological material may beimmobilized to a surface via a gel.

In some implementations, nucleic acids may be immobilized to a surfaceand amplified using bridge amplification. Another useful method foramplifying nucleic acids on a surface is rolling circle amplification(RCA), for example, using methods set forth in further detail below. Insome implementations, the nucleic acids may be attached to a surface andamplified using one or more primer pairs. For example, one of theprimers may be in solution and the other primer may be immobilized onthe surface (e.g., 5′-attached). By way of example, a nucleic acidmolecule may hybridize to one of the primers on the surface followed byextension of the immobilized primer to produce a first copy of thenucleic acid. The primer in solution then hybridizes to the first copyof the nucleic acid which may be extended using the first copy of thenucleic acid as a template. Optionally, after the first copy of thenucleic acid is produced, the original nucleic acid molecule mayhybridize to a second immobilized primer on the surface and may beextended at the same time or after the primer in solution is extended.In any implementation, repeated rounds of extension (e.g.,amplification) using the immobilized primer and primer in solution maybe used to provide multiple copies of the nucleic acid. In someimplementations, the biological material may be confined within apredetermined space with reaction components that are configured to beused during amplification of the biological material (e.g., PCR).

One or more implementations set forth herein may be configured toexecute an assay protocol that is or includes an amplification (e.g.,PCR) protocol. During the amplification protocol, a temperature of thebiological material within a reservoir or channel may be changed inorder to amplify a target sequence or the biological material (e.g., DNAof the biological material). By way of example, the biological materialmay experience (1) a pre-heating stage of about 95° C. for about 75seconds; (2) a denaturing stage of about 95° C. for about 15 seconds;(3) an annealing-extension stage of about of about 59° C. for about 45seconds; and (4) a temperature holding stage of about 72° C. for about60 seconds. Implementations may execute multiple amplification cycles.It is noted that the above cycle describes only one particularimplementation and that alternative implementations may includemodifications to the amplification protocol.

The methods and systems set forth herein may use arrays having featuresat any of a variety of densities including, for example, at least about10 features/cm², about 100 features/cm², about 500 features/cm², about1,000 features/cm², about 5,000 features/cm², about 10,000 features/cm²,about 50,000 features/cm², about 100,000 features/cm², about 1,000,000features/cm², about 5,000,000 features/cm², or higher. The methods andapparatus set forth herein may include detection components or deviceshaving a resolution that is at least sufficient to resolve individualfeatures at one or more of these densities.

The base instrument 102 may include a user interface 130 that isconfigured to receive user inputs for conducting a designated assayprotocol and/or configured to communicate information to the userregarding the assay. The user interface 130 may be incorporated with thebase instrument 102. For example, the user interface 130 may include atouchscreen that is attached to a housing of the base instrument 102 andconfigured to identify a touch from the user and a location of the touchrelative to information displayed on the touchscreen. Alternatively, theuser interface 130 may be located remotely with respect to the baseinstrument 102.

II. Cartridge

The removable cartridge 200 is configured to separably engage orremovably couple to the base instrument 102 at a cartridge chamber 140.As used herein, when the terms “separably engaged” or “removablycoupled” (or the like) are used to describe a relationship between aremovable cartridge 200 and a base instrument 102. The term is intendedto mean that a connection between the removable cartridge 200 and thebase instrument 102 are separable without destroying the base instrument102. Accordingly, the removable cartridge 200 may be separably engagedto the base instrument 102 in an electrical manner such that theelectrical contacts of the base instrument 102 are not destroyed. Theremovable cartridge 200 may be separably engaged to the base instrument102 in a mechanical manner such that features of the base instrument 102that hold the removable cartridge 200, such as the cartridge chamber140, are not destroyed. The removable cartridge 200 may be separablyengaged to the base instrument 102 in a fluidic manner such that theports of the base instrument 102 are not destroyed. The base instrument102 is not considered to be “destroyed,” for example, if only a simpleadjustment to the component (e.g., realigning) or a simple replacement(e.g., replacing a nozzle) is required. Components (e.g., the removablecartridge 200 and the base instrument 102) may be readily separable whenthe components may be separated from each other without undue effort ora significant amount of time spent in separating the components. In someimplementations, the removable cartridge 200 and the base instrument 102may be readily separable without destroying either the removablecartridge 200 or the base instrument 102.

In some implementations, the removable cartridge 200 may be permanentlymodified or partially damaged during a session with the base instrument102. For instance, containers holding liquids may include foil coversthat are pierced to permit the liquid to flow through the system 100. Insuch implementations, the foil covers may be damaged such that thedamaged container is to be replaced with another container. Inparticular implementations, the removable cartridge 200 is a disposablecartridge such that the removable cartridge 200 may be replaced andoptionally disposed after a single use. Similarly, a flow cell of theremovable cartridge 200 may be separately disposable such that the flowcell may be replaced and optionally disposed after a single use.

In other implementations, the removable cartridge 200 may be used formore than one session while engaged with the base instrument 102 and/ormay be removed from the base instrument 102, reloaded with reagents, andre-engaged to the base instrument 102 to conduct additional designatedreactions. Accordingly, the removable cartridge 200 may be refurbishedin some cases such that the same removable cartridge 200 may be usedwith different consumables (e.g., reaction components and biologicalmaterials). Refurbishing may be carried out at a manufacturing facilityafter the cartridge 200 has been removed from a base instrument 102located at a customer's facility.

The cartridge chamber 140 may include a slot, mount, connectorinterface, and/or any other feature to receive the removable cartridge200 or a portion thereof to interact with the base instrument 102.

The removable cartridge 200 may include a fluidic network that may holdand direct fluids (e.g., liquids or gases) therethrough. The fluidicnetwork may include a plurality of interconnected fluidic elements thatare capable of storing a fluid and/or permitting a fluid to flowtherethrough. Non-limiting examples of fluidic elements includechannels, ports of the channels, cavities, storage modules, reservoirsof the storage modules, reaction chambers, waste reservoirs, detectionchambers, multipurpose chambers for reaction and detection, and thelike. For example, the consumable reagent portion 210 may include one ormore reagent wells or chambers storing reagents and may be part of orcoupled to the fluidic network. The fluidic elements may be fluidicallycoupled to one another in a designated manner so that the system 100 iscapable of performing sample preparation and/or analysis.

As used herein, the term “fluidically coupled” (or like term) refers totwo spatial regions being connected together such that a liquid or gasmay be directed between the two spatial regions. In some cases, thefluidic coupling permits a fluid to be directed back and forth betweenthe two spatial regions. In other cases, the fluidic coupling isuni-directional such that there is only one direction of flow betweenthe two spatial regions. For example, an assay reservoir may befluidically coupled with a channel such that a liquid may be transportedinto the channel from the assay reservoir. However, in someimplementations, it may not be possible to direct the fluid in thechannel back to the assay reservoir. In particular implementations, thefluidic network may be configured to receive a biological material anddirect the biological material through sample preparation and/or sampleanalysis. The fluidic network may direct the biological material andother reaction components to a waste reservoir.

FIG. 2 depicts an implementation of a consumable cartridge 300. Theconsumable cartridge may be part of a combined removable cartridge, suchas consumable reagent portion 210 of removable cartridge 200 of FIG. 1;or may be a separate reagent cartridge. The consumable cartridge 300 mayinclude a housing 302 and a top 304. The housing 302 may comprise anon-conductive polymer or other material and be formed to make one ormore reagent chambers 310, 320, 330. The reagent chambers 310, 320, 330may be varying in size to accommodate varying volumes of reagents to bestored therein. For instance, a first chamber 310 may be larger than asecond chamber 320, and the second chamber 320 may be larger than athird chamber 330. The first chamber 310 is sized to accommodate alarger volume of a particular reagent, such as a buffer reagent. Thesecond chamber 320 may be sized to accommodate a smaller volume ofreagent than the first chamber 310, such as a reagent chamber holding acleaving reagent. The third chamber 330 may be sized to accommodate aneven smaller volume of reagent than the first chamber 310 and the secondchamber 320, such as a reagent chamber holding a fully functionalnucleotide containing reagent.

In the illustrated implementation, the housing 302 has a plurality ofhousing walls or sides 350 forming the chambers 310, 320, 330 therein.In the illustrated implementation, the housing 302 forms a structurethat is at least substantially unitary or monolithic. In alternativeimplementations, the housing 302 may be constructed by one or moresub-components that are combined to form the housing 302, such asindependently formed compartments for chambers 310, 320, and 330.

The housing 302 may be sealed by the top 304 once reagents are providedinto the respective chambers 310, 320, 330. The top 304 may comprise aconductive or non-conductive material. For instance, the top 304 may bean aluminum foil seal that is adhesively coupled to top surfaces of thehousing 302 to seal the reagents within their respective chambers 310,320, 330. In other implementations, the top 304 may be a plastic sealthat is adhesively coupled to top surfaces of the housing 302 to sealthe reagents within their respective chambers 310, 320, 330.

In some implementations, the housing 302 may also include an identifier390. The identifier 390 may be a radio-frequency identification (RFID)transponder, a barcode, an identification chip, and/or other identifier.In some implementations, the identifier 390 may be embedded in thehousing 302 or attached to an exterior surface. The identifier 390 mayinclude data for a unique identifier for the consumable cartridge 300and/or data for a type of the consumable cartridge 300. The data of theidentifier 390 may be read by the base instrument 102 or a separatedevice configured for warming the consumable cartridge 300, as describedherein.

In some implementations, the consumable cartridge 300 may include othercomponents, such as valves, pumps, fluidic lines, ports, etc. In someimplementations, the consumable cartridge 300 may be contained within afurther exterior housing.

III. System Controller

The base instrument 102 may also include a system controller 120 that isconfigured to control operation of at least one of the removablecartridge 200 and/or the detection assembly 110. The system controller120 may be implemented utilizing any combination of dedicated hardwarecircuitry, boards, DSPs, processors, etc. Alternatively, the systemcontroller 120 may be implemented utilizing an off-the-shelf PC with asingle processor or multiple processors, with the functional operationsdistributed between the processors. As a further option, the systemcontroller 120 may be implemented utilizing a hybrid configuration inwhich certain modular functions are performed utilizing dedicatedhardware, while the remaining modular functions are performed utilizingan off-the-shelf PC and the like.

The system controller 120 may include a plurality of circuitry modulesthat are configured to control operation of certain components of thebase instrument 102 and/or the removable cartridge 200. The term“module” herein may refer to a hardware device configured to performspecific task(s). For instance, the circuitry modules may include aflow-control module that is configured to control flow of fluids throughthe fluidic network of the removable cartridge 200. The flow-controlmodule may be operably coupled to valve actuators and/or s system pump.The flow-control module may selectively activate the valve actuatorsand/or the system pump to induce flow of fluid through one or more pathsand/or to block flow of fluid through one or more paths.

The system controller 120 may also include a thermal-control module. Thethermal-control module may control a thermocycler or other thermalcomponent to provide and/or remove thermal energy from asample-preparation region of the removable cartridge 200 and/or anyother region of the removeable cartridge 200. In one particular example,a thermocycler may increase and/or decrease a temperature that isexperienced by the biological material in accordance with a PCRprotocol.

The system controller 120 may also include a detection module that isconfigured to control the detection assembly 110 to obtain dataregarding the biological material. The detection module may controloperation of the detection assembly 110 either through a direct wiredconnection or through the contact array if the detection assembly 110 ispart of the removable cartridge 200. The detection module may controlthe detection assembly 110 to obtain data at predetermined times or forpredetermined time periods. By way of example, the detection module maycontrol the detection assembly 110 to capture an image of a reactionchamber of the flow cell receiving portion 220 of the removablecartridge when the biological material has a fluorophore attachedthereto. In some implementations, a plurality of images may be obtained.

Optionally, the system controller 120 may include an analysis modulethat is configured to analyze the data to provide at least partialresults to a user of the system 100. For example, the analysis modulemay analyze the imaging data provided by the detection assembly 110. Theanalysis may include identifying a sequence of nucleic acids of thebiological material.

The system controller 120 and/or the circuitry modules described abovemay include one or more logic-based devices, including one or moremicrocontrollers, processors, reduced instruction set computers (RISC),application specific integrated circuits (ASICs), field programmablegate array (FPGAs), logic circuits, and any other circuitry capable ofexecuting functions described herein. In an implementation, the systemcontroller 120 and/or the circuitry modules execute a set ofinstructions that are stored in a computer- or machine-readable mediumtherein in order to perform one or more assay protocols and/or otheroperations. The set of instructions may be stored in the form ofinformation sources or physical memory elements within the baseinstrument 102 and/or the removable cartridge 200. The protocolsperformed by the system 100 may be used to carry out, for example,machine-writing DNA or otherwise synthesizing DNA (e.g., convertingbinary data into a DNA sequence and then synthesizing DNA strands orother polynucleotides representing the binary data), quantitativeanalysis of DNA or RNA, protein analysis, DNA sequencing (e.g.,sequencing-by-synthesis (SBS)), sample preparation, and/or preparationof fragment libraries for sequencing.

The set of instructions may include various commands that instruct thesystem 100 to perform specific operations such as the methods andprocesses of the various implementations described herein. The set ofinstructions may be in the form of a software program. As used herein,the terms “software” and “firmware” are interchangeable and include anycomputer program stored in memory for execution by a computer, includingRAM memory, ROM memory, EPROM memory, EEPROM memory, and non-volatileRAM (NVRAM) memory. The above memory types are only examples and arethus not limiting as to the types of memory usable for storage of acomputer program.

The software may be in various forms such as system software orapplication software. Further, the software may be in the form of acollection of separate programs, or a program module within a largerprogram or a portion of a program module. The software also may includemodular programming in the form of object-oriented programming. Afterobtaining the detection data, the detection data may be automaticallyprocessed by the system 100, processed in response to user inputs, orprocessed in response to a request made by another processing machine(e.g., a remote request through a communication link).

The system controller 120 may be connected to the other components orsub-systems of the system 100 via communication links, which may behardwired or wireless. The system controller 120 may also becommunicatively connected to off-site systems or servers. The systemcontroller 120 may receive user inputs or commands, from a userinterface 130. The user interface 130 may include a keyboard, mouse, atouch-screen panel, and/or a voice recognition system, and the like.

The system controller 120 may serve to provide processing capabilities,such as storing, interpreting, and/or executing software instructions,as well as controlling the overall operation of the system 100. Thesystem controller 120 may be configured and programmed to control dataand/or power aspects of the various components. Although the systemcontroller 120 is represented as a single structure in FIG. 1, it isunderstood that the system controller 120 may include multiple separatecomponents (e.g., processors) that are distributed throughout the system100 at different locations. In some implementations, one or morecomponents may be integrated with the base instrument 102 and one ormore components may be located remotely with respect to the baseinstrument 102.

IV. Flow Cell

FIGS. 3-4 depict an example of a flow cell 400 that may be used withsystem 100. Flow cell of this example includes a body defining aplurality of elongate flow channels 410, which are recessed below anupper surface 404 of the body 402. The flow channels 410 are generallyparallel with each other and extend along substantially the entirelength of body 402. While five flow channels 410 are shown, a flow cell400 may include any other suitable number of flow channels 410,including more or fewer than five flow channels 410. The flow cell 400of this example also includes a set of inlet ports 420 and a set ofoutlet ports 422, with each port 420, 422 being associated with acorresponding flow channel 410. Thus, each inlet port 420 may beutilized to communicate fluids (e.g., reagents, etc.) to thecorresponding channel 410; while each outlet port 422 may be utilized tocommunicate fluids from the corresponding flow channel 410.

In some versions, the flow cell 400 is directly integrated into the flowcell receiving portion 220 of the removable cartridge 200. In some otherversions, the flow cell 400 is removably coupled with the flow cellreceiving portion 220 of the removable cartridge 200. In versions wherethe flow cell 400 is either directly integrated into the flow cellreceiving portion 220 or removably coupled with the flow cell receivingportion 220, the flow channels 410 of the flow cell 400 may receivefluids from the consumable reagent portion 210 via the inlet ports 420,which may be fluidly coupled with reagents stored in the consumablereagent portion 210. Of course, the flow channels 410 may be coupledwith various other fluid sources or reservoirs, etc., via the ports 420,422. As another illustrative variation, some versions of consumablecartridge 300 may be configured to removably receive or otherwiseintegrate the flow cell 400. In such versions, the flow channels 410 ofthe flow cell 400 may receive fluids from the reagent chambers 310, 320,330 via the inlet ports 420. Other suitable ways in which the flow cell400 may be incorporated into the system 100 will be apparent to thoseskilled in the art in view of the teachings herein.

FIG. 4 shows a flow channel 410 of the flow cell 400 in greater detail.As shown, the flow channel 410 includes a plurality of wells 430 formedin a base surface 412 of the flow channel 410. As will be described ingreater detail below each well 430 is configured to contain DNA strandsor other polynucleotides, such as machine-written polynucleotides. Insome versions, each well 430 has a cylindraceous configuration, with agenerally circular cross-sectional profile. In some other versions, eachwell 430 has a polygonal (e.g., hexagonal, octagonal, etc.)cross-sectional profile. Alternatively, wells 430 may have any othersuitable configuration. It should also be understood that wells 430 maybe arranged in any suitable pattern, including but not limited to a gridpattern.

FIG. 5 shows a portion of a channel within a flow cell 500 that is anexample of a variation of the flow cell 400. In other words, the channeldepicted in FIG. 5 is a variation of the flow channel 410 of the flowcell 400. This flow cell 500 is operable to read polynucleotide strands550 that are secured to the floor 534 of wells 530 in the flow cell 500.By way of example only, the floor 534 where polynucleotide strands 550are secured may include a co-block polymer capped with azido. By way offurther example only, such a polymer may comprise apoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM)coating provided in accordance with at least some of the teachings ofU.S. Pat. No. 9,012,022, entitled “Polymer Coatings,” issued Apr. 21,2015, which is incorporated by reference herein in its entirety. Such apolymer may be incorporated into any of the various flow cells describedherein.

In the present example, the wells 530 are separated by interstitialspaces 514 provided by the base surface 512 of the flow cell 500. Eachwell 530 has a sidewall 532 and a floor 534. The flow cell 500 in thisexample is operable to provide an image sensor 540 under each well 530.In some versions, each well 530 has at least one corresponding imagesensor 540, with the image sensors 540 being fixed in position relativeto the wells 530. Each image sensor 540 may comprise a CMOS imagesensor, a CCD image sensor, or any other suitable kind of image sensor.By way of example only, each well 530 may have one associated imagesensor 540 or a plurality of associated image sensors 540. As anothervariation, a single image sensor 540 may be associated with two or morewells 530. In some versions, one or more image sensors 540 move relativeto the wells 530, such that a single image sensor 540 or single group ofimage sensors 540 may be moved relative to the wells 530. As yet anothervariation, the flow cell 500 may be movable in relation to the singleimage sensor 540 or single group of image sensors 540, which may be atleast substantially fixed in position.

Each image sensor 540 may be directly incorporated into the flow cell500. Alternatively, each image sensor 540 may be directly incorporatedinto a cartridge such as the removable cartridge 200, with the flow cell500 being integrated into or otherwise coupled with the cartridge. Asyet another illustrative variation, each image sensor 540 may bedirectly incorporated into the base instrument 102 (e.g., as part of thedetection assembly 110 noted above). Regardless of where the imagesensor(s) 540 is/are located, the image sensor(s) 540 may be integratedinto a printed circuit that includes other components (e.g., controlcircuitry, etc.). In versions where the one or more image sensors 540are not directly incorporated into the flow cell 500, the flow cell 500may include optically transmissive features (e.g., windows, etc.) thatallow the one or more image sensors 540 to capture fluorescence emittedby the one or more fluorophores associated with the polynucleotidestrands 550 that are secured to the floors 534 of the wells 530 in theflow cell 500 as described in greater detail below. It should also beunderstood that various kinds of optical elements (e.g., lenses, opticalwaveguides, etc.) may be interposed between the floors 534 of the wells530 and the corresponding image sensor(s) 540.

As also shown in FIG. 5, a light source 560 is operable to project light562 into the well 530. In some versions, each well 530 has at least onecorresponding light source 560, with the light sources 560 being fixedin position relative to the wells 530. By way of example only, each well530 may have one associated light source 560 or a plurality ofassociated light sources 560. As another variation, a single lightsource 560 may be associated with two or more wells 530. In some otherversions, one or more light sources 560 move relative to the wells 530,such that a single light source 560 or single group of light sources 560may be moved relative to the wells 530. As yet another variation, theflow cell 500 may be movable in relation to the single light source 560or single group of light sources 560, which may be substantially fixedin position. By way of example only, each light source 560 may includeone or more lasers. In another example, the light source 560 may includeone or more diodes.

Each light source 560 may be directly incorporated into the flow cell500. Alternatively, each light source 560 may be directly incorporatedinto a cartridge such as the removable cartridge 200, with the flow cell500 being integrated into or otherwise coupled with the cartridge. Asyet another illustrative variation, each light source 560 may bedirectly incorporated into the base instrument 102 (e.g., as part of thedetection assembly 110 noted above). In versions where the one or morelight sources 560 are not directly incorporated into the flow cell 500,the flow cell 500 may include optically transmissive features (e.g.,windows, etc.) that allow the wells 530 to receive the light emitted bythe one or more light source 560, to thereby enable the light to reachthe polynucleotide strands 550 that are secured to the floor 534 of thewells 530. It should also be understood that various kinds of opticalelements (e.g., lenses, optical waveguides, etc.) may be interposedbetween the wells 530 and the corresponding light source(s) 560.

As described elsewhere herein and as is shown in block 590 of FIG. 6, aDNA reading process may begin with performing a sequencing reaction inthe targeted well(s) 530 (e.g., in accordance with at least some of theteachings of U.S. Pat. No. 9,453,258, entitled “Methods and Compositionsfor Nucleic Acid Sequencing,” issued Sep. 27, 2016, which isincorporated by reference herein in its entirety). Next, as shown inblock 592 of FIG. 6, the light source(s) 560 is/are activated over thetargeted well(s) 530 to thereby illuminate the targeted well(s) 530. Theprojected light 562 may cause a fluorophore associated with thepolynucleotide strands 550 to fluoresce. Accordingly, as shown in block594 of FIG. 6, the corresponding image sensor(s) 540 may detect thefluorescence emitted from the one or more fluorophores associated withthe polynucleotide strands 550. The system controller 120 of the baseinstrument 102 may drive the light source(s) 560 to emit the light. Thesystem controller 120 of the base instrument 102 may also process theimage data obtained from the image sensor(s) 540, representing thefluorescent emission profiles from the polynucleotide strands 550 in thewells 530. Using this image data from the image sensor(s) 540, and asshown in block 596 of FIG. 6, the system controller 120 may determinethe sequence of bases in each polynucleotide strand 550. By way ofexample only, this process and equipment may be utilized to map a genomeor otherwise determine biological information associated with anaturally occurring organism, where DNA strands or other polynucleotidesare obtained from or otherwise based on a naturally occurring organism.Alternatively, the above-described process and equipment may be utilizedto obtain data stored in machine-written DNA as will be described ingreater detail below.

By way of further example only, when carrying out the above-describedprocedure shown in FIG. 6, time space sequencing reactions may utilizeone or more chemistries and imaging events or steps to differentiatebetween a plurality of analytes (e.g., four nucleotides) that areincorporated into a growing nucleic acid strand during a sequencingreaction; or alternatively, fewer than four different colors may bedetected in a mixture having four different nucleotides while stillresulting in the determination of the four different nucleotides (e.g.,in a sequencing reaction). A pair of nucleotide types may be detected atthe same wavelength, but distinguished based on a difference inintensity for one member of the pair compared to the other, or based ona change to one member of the pair (e.g., via chemical modification,photochemical modification, or physical modification) that causesapparent signal to appear or disappear compared to the signal detectedfor the other member of the pair.

V. Machine-Writing Biological Material

In some implementations, a system 100 such as the system 100 shown inFIG. 1 may be configured to synthesize biological materials (e.g.polynucleotide, such as DNA) to encode data that may later be retrievedthrough the performance of assays such as those described above. In someimplementations, this type of encoding may be performed by assigningvalues to nucleotide bases (e.g., binary values, such as 0 or 1, ternaryvalues such as 0, 1 or 2, etc.), converting the data to be encoded intoa string of the relevant values (e.g., converting a textual message intoa binary string using the ASCII encoding scheme), and then creating oneor more polynucleotides made up of nucleotides having bases in asequence corresponding to the string obtained by converting the data.

In some implementations, the creation of such polynucleotides may beperformed using a version of the flow cell 400 having an array of wells630 that are configured as shown in FIG. 7. FIG. 7 shows a portion of achannel within a flow cell 600 that is an example of a variation of theflow cell 400. In other words, the channel depicted in FIG. 7 is avariation of the flow channel 410 of the flow cell 400. In this example,each well 630 is recessed below a base surface 612 of the flow cell 600.The wells 630 are thus spaced apart from each other by interstitialspaces 614. By way of example only, the wells 630 may be arranged in agrid or any other suitable pattern along the base surface 612 of theflow cell 600. Each well 630 of this example includes a sidewall 632 anda floor 634. Each well 630 of this example further includes a respectiveelectrode assembly 640 positioned on the floor 634 of the well 630. Insome versions, each electrode assembly 640 includes just a singleelectrode element. In some other versions, each electrode assembly 640includes a plurality of electrode elements or segments. The terms“electrode” and “electrode assembly” should be read herein as beinginterchangeable.

Base instrument 102 is operable to independently activate electrodeassemblies 640, such that one or more electrode assemblies 640 may be inan activated state while one or more other electrode assemblies 640 arenot in an activated state. In some versions, a CMOS device or otherdevice is used to control electrode assemblies 640. Such a CMOS devicemay be integrated directly into the flow cell 600, may be integratedinto a cartridge (e.g., cartridge 200) in which the flow cell 600 isincorporated, or may be integrated directly into the base instrument102. As shown in FIG. 7, each electrode assembly 640 extends along thefull width of floor 634, terminating at the sidewall 632 of thecorresponding well 630. In other versions, each electrode assembly 640may extend along only a portion of the floor 634. For instance, someversions of electrode assembly 640 may terminate interiorly relative tothe sidewall 632. While each electrode assembly 540 is schematicallydepicted as a single element in FIG. 5, it should be understood thateach electrode assembly 540 may in fact be formed by a plurality ofdiscrete electrodes rather than just consisting of one single electrode.

As shown in FIG. 7, specific polynucleotide strands 650 may be createdin individual wells 630 by activating the electrode assembly 640 of therelevant wells 630 to electrochemically generate acid that may deprotectthe end group of the polynucleotide strand 650 in the well 630. By wayof example only, polynucleotide strands 650 may be chemically attachedto the surface at the bottom of the well 630 using linkers havingchemistries such as silane chemistry on one end and DNA synthesiscompatible chemistry (e.g., a short oligo for enzyme to bind to) on theother end.

To facilitate reagent exchange (e.g., transmission of a deblockingagent), each electrode assembly 640 and the floor 634 of each well 630may include at least one opening 660 in this example. The openings 660may be fluidly coupled with a flow channel 662 that extends underneaththe wells 630, below the floors 634. To provide such an opening 660through the electrode assembly 640, the electrode assembly 640 may beannular in shape, may be placed in quadrants, may be placed on theperimeter or sidewall 632 of the well 630, or may be placed or shaped inother suitable manners to avoid interference with reagent exchangeand/or passage of light (e.g., as may be used in a sequencing processthat involved detection of fluorescent emissions). In otherimplementations, reagents may be provided into the flow channel of theflow cell 600 without the openings 660. It should be understood that theopenings 660 may be optional and may be omitted in some versions.Similarly, the flow channel 662 may be optional and may be omitted insome versions.

FIG. 9 shows an example of a form that electrode assembly 640 may take.In this example, electrode assembly 640 includes four discrete electrodesegments 642, 644, 646, 648 that together define an annular shape. Theelectrode segments 642, 644, 646, 648 are thus configured as discreteyet adjacent quadrants of a ring. Each electrode segment 642, 644, 646,648 may be configured to provide a predetermined charge that is uniquelyassociated with a particular nucleotide. For instance, electrode segment642 may be configured to provide a charge that is uniquely associatedwith adenine; electrode segment 644 may be configured to provide acharge that is uniquely associated with cytosine; electrode segment 646may be configured to provide a charge that is uniquely associated withguanine; and electrode segment 648 may be configured to provide a chargethat is uniquely associated with thymine. When a mixture of those fournucleotides are flowed through the flow channel above the wells 630,activation of electrode segments 642, 644, 646, 648 may cause thecorresponding nucleotides from that flow to adhere to the strand 650.Thus, when electrode segment 642 is activated, it may effect writing ofadenine to the strand 650; when electrode segment 644 is activated, itmay effect writing of cytosine to the strand 650; when electrode segment646 is activated, it may effect writing of guanine to the strand 650;and when electrode segment 648 is activated, it may effect writing ofthymine to the strand 650. This writing may be provided by the activatedelectrode segment 642, 644, 646, 648 hybridizing the inhibitor of theenzyme for the pixel associated with the activated electrode segment642, 644, 646, 648. While electrode segments 642, 644, 646, 648 areshown as forming an annular shape in FIG. 9, it should be understoodthat any other suitable shape or shapes may be formed by electrodesegments 642, 644, 646, 648. In still other implementations, a singleelectrode may be utilized for the electrode assembly 640 and the chargemay be modulated to incorporate various nucleotides to be written to theDNA strand or other polynucleotide.

As another example, the electrode assembly 640 may be activated toprovide a localized (e.g., localized within the well 630 in which theelectrode assembly 640 is disposed), electrochemically generated changein pH; and/or electrochemically generate a moiety (e.g., a reducing oroxidizing reagent) locally to remove a block from a nucleotide. As yetanother variation, different nucleotides may have different blocks; andthose blocks may be photocleaved based on a wavelength of lightcommunicated to the well 630 (e.g., light 562 projected from the lightsource 560). As still another variation, different nucleotides may havedifferent blocks; and those blocks may be cleaved based on certain otherconditions. For instance, one of the four blocks may be removed based ona combination of a reducing condition plus either high local pH or lowlocal pH; another of the four blocks may be removed based on acombination of an oxidative condition plus either high local pH or lowlocal pH; another of the four blocks may be removed based on acombination of light and a high local pH; and another of the four blocksmay be removed based on a combination of light and a low local pH. Thus,four nucleotides may be incorporated at the same time, but withselective unblocking occurring in response to four different sets ofconditions.

The electrode assembly 640 further defines the opening 660 at the centerof the arrangement of the electrode segments 642, 644, 646, 648. Asnoted above, this opening 660 may provide a path for fluid communicationbetween the flow channel 662 and the wells 630, thereby allowingreagents, etc. that are flowed through the flow channel 662 to reach thewells 630. As also noted above, some variations may omit the flowchannel 662 and provide communication of reagents, etc. to the wells 630in some other fashion (e.g., through passive diffusion, etc.).Regardless of whether fluid is communicated through the opening 660, theopening 660 may provide a path for optical transmission through thebottom of the well 630 during a read cycle, as described herein. In someversions, the opening 660 may be optional and may thus be omitted. Inversions where the opening 660 is omitted, fluids may be communicated tothe wells 630 via one or more flow channels that are above the wells 630or otherwise positioned in relation to the wells 630. Moreover, theopening 660 may not be needed for providing a path for opticaltransmission through the bottom of the well 630 during a read cycle. Forinstance, as described below in relation to the flow cell 601, theelectrode assembly 640 may comprise an optically transparent material(e.g., optically transparent conducting film (TCF), etc.), and the flowcell 600 itself may comprise an optically transparent material (e.g.,glass), such that the electrode assembly 640 and the material formingthe flow cell 600 may allow the fluorescence emitted from the one ormore fluorophores associated with the machine-written polynucleotidestrands 650 to reach an image sensor 540 that is under the well 630.

FIG. 8 shows an example of a process that may be utilized in the flowcell 600 to machine-write polynucleotides or other nucleotide sequences.At the beginning of the process, as shown in the first block 690 of FIG.8, nucleotides may be flowed into the flow cell 600, over the wells 630.As shown in the next block 692 in FIG. 8, the electrode assembly 640 maythen be activated to write a first nucleotide to a primer at the bottomof a targeted well 630. As shown in the next block 694 of FIG. 8, aterminator may then be cleaved off the first nucleotide that was justwritten in the targeted well 630. Various suitable ways in which aterminator may be cleaved off the first nucleotide will be apparent tothose skilled in the art in view of the teachings herein. Once theterminator is cleaved off the first nucleotide, as shown in the nextblock 696 of FIG. 8, the electrode assembly 640 may be activated towrite a second nucleotide to the first nucleotide. While not shown inFIG. 8, a terminator may be cleaved off the second nucleotide, then athird nucleotide may be written to the second nucleotide, and so onuntil the desired sequence of nucleotides has been written.

In some implementations, encoding of data via synthesis of biologicalmaterials such as DNA may be performed in other manners. For example, insome implementations, the flow cell 600 may lack the electrode assembly640 altogether. For instance, deblock reagents may be selectivelycommunicated from the flow channel 662 to the wells 630 through theopenings 660. This may eliminate the need for electrode assemblies 640to selectively activate nucleotides. As another example, an array ofwells 630 may be exposed to a solution containing all nucleotide basesthat may be used in encoding the data, and then individual nucleotidesmay be selectively activated for individual wells 630 by using lightfrom a spatial light modulator (SLM). As another example, in someimplementations individual bases may be assigned combined values (e.g.,adenine may be used to encode the binary couplet 00, guanine may be usedto encode the binary couplet 01, cytosine may be used to encode thebinary couplet 10, and thymine may be used to encode the binary couplet11) to increase the storage density of the polynucleotides beingcreated. Other examples are also possible and will be immediatelyapparent to those skilled in the art in light of this disclosure.Accordingly, the above description of synthesizing biological materialssuch as DNA to encode data should be understood as being illustrativeonly; and should not be treated as limiting.

VI. Reading Machine-Written Biological Material

After polynucleotide strands 650 have been machine-written in one ormore wells 630 of a flow cell 600, the polynucleotide strands 650 may besubsequently read to extract whatever data or other information wasstored in the machine-written polynucleotide strands 650. Such a readingprocess may be carried out using an arrangement such as that shown inFIG. 5 and described above. In other words, one or more light sources560 may be used to illuminate one or more fluorophores associated withthe machine-written polynucleotide strands 650; and one or more imagesensors 540 may be used to detect the fluorescent light emitted by theilluminated one or more fluorophores associated with the machine-writtenpolynucleotide strands 650. The fluorescence profile of the lightemitted by the illuminated one or more fluorophores associated with themachine-written polynucleotide strands 650 may be processed to determinethe sequence of bases in the machine-written polynucleotide strands 650.This determined sequence of bases in the machine-written polynucleotidestrands 650 may be processed to determine the data or other informationthat was stored in the machine-written polynucleotide strands 650.

In some versions, the machine-written polynucleotide strands 650 remainin the flow cell 600 containing wells 630 for a storage period. When itis desired to read the machine-written polynucleotide strands 650, theflow cell 600 may permit the machine-written polynucleotide strands 650to be read directly from the flow cell. By way of example only, the flowcell 600 containing wells 630 may be received in a cartridge (e.g.,cartridge 200) or base instrument 102 containing light sources 560and/or image sensors 540, such that the machine-written polynucleotidestrands 650 are read directly from the wells 630.

As another illustrative example, the flow cell containing wells 630 maydirectly incorporate one or both of light source(s) 560 or imagesensor(s) 540. FIG. 10 shows an example of a flow cell 601 that includeswells 630 with electrode assemblies 640, one or more image sensors 540,and a control circuit 670. Like in the flow cell 500 depicted in FIG. 5,the flow cell 601 of this example is operable to receive light 562projected from a light source 560. This projected light 562 may causeone or more fluorophores associated with the machine-writtenpolynucleotide strands 650 to fluoresce; and the corresponding imagesensor(s) 540 may capture the fluorescence emitted from the one or morefluorophores associated with the machine-written polynucleotide strands650.

As noted above in the context of the flow cell 500, each well 650 of theflow cell 601 may include its own image sensor 540 and/or its own lightsource 560; or these components may be otherwise configured and arrangedas described above. In the present example, the fluorescence emittedfrom the one or more fluorophores associated with the machine-writtenpolynucleotide strands 650 may reach the image sensor 540 via theopening 660. In addition, or in the alternative, the electrode assembly640 may comprise an optically transparent material (e.g., opticallytransparent conducting film (TCF), etc.), and the flow cell 601 itselfmay comprise an optically transparent material (e.g., glass), such thatthe electrode assembly 640 and the material forming the flow cell 601may allow the fluorescence emitted from the one or more fluorophoresassociated with machine-written polynucleotide strands 650 to reach theimage sensor 540. Moreover, various kinds of optical elements (e.g.,lenses, optical waveguides, etc.) may be interposed between the wells650 and the corresponding image sensor(s) to ensure that the imagesensor 540 is only receiving fluorescence emitted from the one or morefluorophores associated with the machine-written polynucleotide strands650 of the desired well(s) 630.

In the present example, the control circuit 670 is integrated directlyinto the flow cell 601. By way of example only, the control circuit 670may comprise a CMOS chip and/or other printed circuitconfigurations/components. The control circuit 670 may be incommunication with the image sensor(s) 540, the electrode assembly(ies)640, and/or the light source 560. In this context, “in communication”means that the control circuit 670 is in electrical communication withimage sensor(s) 540, the electrode assembly(ies) 640, and/or the lightsource 560. For instance, the control circuit 670 may be operable toreceive and process signals from the image sensor(s) 540, with thesignals representing images that are picked up by the image sensor(s)540. “In communication” in this context may also include the controlcircuit 670 providing electrical power to the image sensor(s) 540, theelectrode assembly(ies) 640, and/or the light source 560.

In some versions, each image sensor 540 has a corresponding controlcircuit 670. In some other versions, a control circuit 670 is coupledwith several, if not all, of the image sensors in the flow cell 601.Various suitable components and configurations that may be used toachieve this will be apparent to those skilled in the art in view of theteachings herein. It should also be understood that the control circuit670 may be integrated, in whole or in part, in a cartridge (e.g.,removable cartridge 200) and/or in the base instrument 102, in additionto or in lieu of being integrated into the flow cell 601.

As still another illustrative example, regardless of whether awrite-only flow cell like the flow cell 600 of FIG. 7 or a read-writeflow cell like the flow cell 601 of FIG. 10 is used, the machine-writtenpolynucleotide strands 650 may be transferred from wells 630 after beingsynthesized. This may occur shortly after the synthesis is complete,right before the machine-written polynucleotide strands 650 are to beread, or at any other suitable time. In such versions, themachine-written polynucleotide strands 650 may be transferred to aread-only flow cell like the flow cell 500 depicted in FIG. 5; and thenbe read in that read-only flow cell 500. Alternatively, any othersuitable devices or processes may be used.

In some implementations, reading data encoded through the synthesis ofbiological materials may be achieved by determining the well(s) 630storing the synthesized strand(s) 650 of interest and then sequencingthose strands 650 using techniques such as those described previously(e.g., sequencing-by-synthesis). In some implementations, to facilitatereading data stored in nucleotide sequences, when data is stored, anindex may be updated with information showing the well(s) 630 where thestrand(s) 650 encoding that data was/were synthesized. For example, whenan implementation of a system 100 configured to synthesize strands 650capable of storing up to 256 bits of data is used to store a one megabit(1,048,576 bit) file, the system controller 120 may perform steps suchas: 1) break the file into 4,096 256 bit segments; 2) identify asequence of 4,096 wells 630 in the flow cell 600, 601 that were notcurrently being used to store data; 3) write the 4,096 segments to the4,096 wells 430, 530; 4) update an index to indicate that the sequencestarting with the first identified well 630 and ending at the lastidentified well 630 was being used to store the file. Subsequently, whena request to read the file was made, the index may be used to identifythe well(s) 630 containing the relevant strand(s) 650, the strand(s) 650from those wells 630 may be sequenced, and the sequences may be combinedand converted into the appropriate encoding format (e.g., binary), andthat combined and converted data may then be returned as a response tothe read request.

In some implementations, reading of data previously encoded viasynthesis of biological materials may be performed in other manners. Forexample, in some implementations, if a file corresponding to 4,096 wells630 was to be written, rather than identifying 4,096 sequential wells630 to write it to, a controller may identify 4,096 wells 630 and thenupdate the index with multiple locations corresponding to the file inthe event that those wells 630 did not form a continuous sequence. Asanother example, in some implementations, rather than identifyingindividual wells 630, a system controller 120 may group wells 630together (e.g., into groups of 128 wells 630), thereby reducing theoverhead associated with storing location data (i.e., by reducing theaddressing requirements from one address per well 630 to one address pergroup of wells 630). As another example, in implementations that storedata reflecting the location of wells 630 where DNA strands or otherpolynucleotides have been synthesized, that data may be stored invarious ways, such as sequence identifiers (e.g., well 1, well 2, well3, etc.) or coordinates (e.g., X and Y coordinates of a well's locationin an array).

As another example, in some implementations, rather than reading strands650 from the wells 630 in which they were synthesized, strands 650 maybe read from other locations. For instance, strands 650 may besynthesized to include addresses, and then cleaved from the wells 630and stored in a tube for later retrieval, during which the includedaddress information may be used to identify the strands 650corresponding to particular files. As another illustrative example, thestrands 650 may be copied off the surface using polymerase and theneluted & stored in tube. Alternatively, the strands 650 may be copied onto a bead using biotinylated oligos hybridized to DNA strands or otherpolynucleotides and capturing extended products on streptavidin beadsthat are dispensed in the wells 630. Other examples are also possibleand will be immediately apparent to those of skill in the art in lightof this disclosure. Accordingly, the above description of retrievingdata encoded through the synthesis of biological materials should beunderstood as being illustrative only; and should not be treated aslimiting.

Implementations described herein may utilize a polymer coating for asurface of a flow cell, such as that described in U.S. Pat. No.9,012,022, entitled “Polymer Coatings,” issued Apr. 21, 2015, which isincorporated by reference herein in its entirety. Implementationsdescribed herein may utilize one or more labelled nucleotides having adetectable label and a cleavable linker, such as those described in U.S.Pat. No. 7,414,116, entitled “Labelled Nucleotide Strands,” issued Aug.19, 2008, which is incorporated by reference herein in its entirety. Forinstance, implementations described herein may utilize a cleavablelinker that is cleavable with by contact with water-soluble phosphinesor water-soluble transition metal-containing catalysts having afluorophore as a detectable label. Implementations described herein maydetect nucleotides of a polynucleotide using a two-channel detectionmethod, such as that described in U.S. Pat. No. 9,453,258, entitled“Methods and Compositions for Nucleic Acid Sequencing,” issued Sep. 27,2016, which is incorporated by reference herein in its entirety. Forinstance, implementations described herein may utilize afluorescent-based SBS method having a first nucleotide type detected ina first channel (e.g., dATP having a label that is detected in the firstchannel when excited by a first excitation wavelength), a secondnucleotide type detected in a second channel (e.g., dCTP having a labelthat is detected in a second channel when excited by a second excitationwavelength), a third nucleotide type detected in both the first andsecond channel (e.g., dTTP having at least one label that is detected inboth channels when excited by the first and/or second excitationwavelength), and a fourth nucleotide type that lacks a label that isnot, or that is minimally, detected in either channel (e.g., dGTP havingno label). Implementations of the cartridges and/or flow cells describedherein may be constructed in accordance with one or more teachingsdescribed in U.S. Pat. No. 8,906,320, entitled “Biosensors forBiological or Chemical Analysis and Systems and Methods for Same,”issued Dec. 9, 2014, which is incorporated by reference herein in itsentirety; U.S. Pat. No. 9,512,422, entitled “Gel Patterned Surfaces,”issued Dec. 6, 2016, which is incorporated by reference herein in itsentirety; U.S. Pat. No. 10,254,225, entitled “Biosensors for Biologicalor Chemical Analysis and Methods of Manufacturing the Same,” issued Apr.9, 2019, which is incorporated by reference herein in its entirety;and/or U.S. Pub. No. 2018/0117587, entitled “Cartridge Assembly,”published May 3, 2018, which is incorporated by reference herein in itsentirety.

VII. Features for Providing Selective Deposition or Activation ofNucleotides During Writing Process in DNA Storage Device

When machine-writing polynucleotides in a DNA storage device, it isdesirable to provide substantial precision in the selection andactivation or deposition of nucleotides in a given polynucleotide. Thisincludes selecting the appropriate nucleotide at the appropriate momentin time; and ensuring that the selected nucleotide gets deposited at theappropriate location at the appropriate moment in time. As describedherein, when performing machine-writing of polynucleotides, a solutioncontaining all four nucleotides may be flowed through a cell; and bycoupling one or more of changes in voltage, changes in pH, or lightsensitivity together, a particular nucleotide in the solution may“activated” such that it attaches to a primer or existing polynucleotidein a well of a flow cell. The following provides several illustrativeexamples of how this process may be carried out with substantialprecision.

A. Flow Cell with pH and Photonic Activation of Nucleotides

In addition to, or in lieu of, using electrodes like those of anelectrode assembly 640 to selectively activate nucleotides to effectmachine-writing of polynucleotides, another option is to utilize changesin pH and/or light to provide selective activation. For instance, allreagents may be provided together, and a reaction may occur only whenthe pH value and light value simultaneously reach certain targets. Somenucleotides may also be photoactivated, without involving the pH toreach a certain target. In addition, or in the alternative, somenucleotides may be activated in response to exposure to certain pHvalues, without involving a particular illumination profile foractivation.

FIG. 11 shows another example of a flow cell 700 that may be used toread and write polynucleotides as described herein, providing writingcapabilities based on changes in pH and/or illumination profiles. Theflow cell 700 of this example includes an upper body portion 702, amiddle body portion 704, and a lower body portion 706. An upper fluidflow channel 764 is defined between the upper and middle body portions802, 804 and is operable to receive a flow of fluid (e.g., a fluidcontaining nucleotide bases, etc.). A lower fluid flow channel 762 isdefined between the middle and lower body portions 704, 706 and isoperable to receive a separate flow of fluid (e.g., a fluid containingdeblocking/deshielding agents, etc.). As with lower fluid flow channel662 described above, lower fluid flow channel 762 of this example may beoptional and may be omitted in some variations.

The flow cell 700 of this example further includes a plurality of wells730 that are formed as recesses in the bottom surface 712 of the upperfluid flow channel 764. These wells 730 are substantially similar to thewells 630 described above. The flow cell 700 further defines a pluralityof interstitial spaces 714 between the wells 730. A pH control feature740 is positioned at the bottom of each well 730. Each well 730 lacks anelectrode assembly in this example. In some other variations, each well730 may include an electrode assembly similar to the electrode assembly640 described above. In such variations, the pH control feature 740 andthe electrode assembly may both be positioned at the bottom of eachwell; one may be positioned at the bottom of the well 730 while theother is positioned at the sidewall of the well 730; or any othersuitable positioning may be used.

The pH control feature 740 of the present example is operable to adjustthe pH level within the well 730. By way of example only, the pH controlfeature may comprise a bubble generator, a set of electrodes, or someother feature. The flow cell 700 may be configured to include one ormore pH control features 740 to simultaneously provide pH levels thatdiffer among different wells 730.

The pH control feature 740 may be selectively activated by a controllerthat is directly integrated into the flow cell 700. By way of exampleonly, such an integrated controller may be incorporated into a CMOSchip. In some such versions, the same CMOS chip or other controller alsocontrols other features of the flow cell 700 (e.g., the light sources722, the image sensors 792, etc.). As another illustrative alternative,the pH control feature 740 may be selectively activated by a controllerthat is directly integrated into a cartridge (e.g., the removablecartridge 200) that receives the flow cell 700. As still anotherillustrative alternative, the pH control feature 700 may be selectivelyactivated by a controller that is directly integrated into the baseinstrument 102. Moreover, components of the controller that selectivelyactivates the pH control feature 740 may be distributed among two ormore of the flow cell 700, a cartridge that receives the flow cell 700,or the base instrument 102. Various suitable components and arrangementsthat may be utilized to provide control of the pH control feature 740will be apparent to those skilled in the art in view of the teachingsherein. Examples of how the pH control feature 740 may be utilizedduring a DNA machine-writing process will be described in greater detailbelow.

In the present example, bottom of each well 730 includes an opening 760providing a pathway for fluid communication between the well 730 and thelower fluid flow channel 762. In some versions, this opening 760includes a valve that is operable to selectively open or close tothereby selectively permit or prevent fluid communication from the lowerfluid flow channel 762 to the corresponding well 730. As noted above,the lower fluid flow channel 762 may be optional. Likewise, the opening760 (and corresponding valve, if any) is also optional. Some variationsof the flow cell 700 may lack the opening 760 at the bottom of each well730.

As described above, light may be utilized to read machine-writtenpolynucleotides within the wells 730. To that end, FIG. 11 shows a setof light sources 722 that are configured to emit light towardcorresponding wells 730. While each well 730 has a corresponding lightsource 722 in this example, other variations may provide a light source722 that spans across more than one well 730. The light sources 722 areall coupled with an upper integrated circuit layer 720 and arepositioned to be flush with the upper surface 724 of the upper flowchannel 726 in this example. Alternatively, the light sources 722 mayhave some other relationship with the upper surface 724 of the upperflow channel 726. The upper integrated circuit layer 720 is operable toselectively drive the light sources 722 independently of each other.

By way of example only, the upper integrated circuit layer 720 mayinclude a CMOS chip. By way of further example only, the light sources722 may include microscopic light emitting diodes (microLEDs) that areintegrated in a CMOS chip that is part of the upper integrated circuitlayer 720. Alternatively, the light sources 720 (regardless of whetherthey include microLEDs) may otherwise be coupled with the upperintegrated circuit layer 720. In some versions, each light source 722for each well 730 consists of a single microLED. In some other versions,each light source 722 for each well 730 consists of an array ofmicroLEDs. Alternatively, any other suitable kind of light source may beused for light sources 722.

The flow cell 700 of the present example further includes a lowerintegrated circuit layer 790 with a plurality of image sensors 792. Byway of example only, the lower integrated circuit layer 790 and imagesensors 792 may be part of a CMOS chip. The lower integrated circuitlayer 790 and image sensors 792 are integrated into the lower bodyportion 706 in this example, with the image sensors 792 being positionedat the lower surface 866 of the lower fluid flow channel 762. In someother variations, the lower integrated circuit layer 790 and imagesensors 792 are integrated into some other component. By way of exampleonly, the lower integrated circuit layer 790 and/or image sensors 792may be integrated into a cartridge (e.g., the removable cartridge 200)that receives the flow cell 700; may be integrated into the baseinstrument 102; or may be integrated in some other component. The lowerintegrated circuit layer 790 and image sensors 792 are in communicationwith each other. In this context, “in communication” means that thelower integrated circuit layer 790 is in electrical communication withthe image sensors 792. For instance, the lower integrated circuit layer790 may be operable to receive and process signals from the imagesensors 792, with the signals representing images that are picked up bythe image sensors 792. “In communication” in this context may alsoinclude the lower integrated circuit layer 790 providing electricalpower to the image sensors 792.

Each image sensor 792 is positioned under a corresponding well 730.Thus, when light source(s) 722 is/are activated to emit light toward thewell(s) 730, the corresponding image sensor(s) 792 is/are configured todetect fluorescence emitted by fluorophores associated withpolynucleotides (e.g., machine-written DNA) contained within the well(s)730. The fluorescent light profile detected by image sensors 792 may beutilized to read the polynucleotides as described herein. In some othervariations of the flow cell 700, the image sensors 792 are omitted. Insome such versions, the flow cell 700 is a “write only” flow cell. Insome other versions where the image sensors 792 are omitted, the imagesensors are provided by some other piece of equipment (e.g., theremovable cartridge 200, the base instrument 102, etc.).

As noted above, nucleotides may be selectively activated or depositedbased on exposure to a medium having a particular pH value and/or basedon exposure to light having a certain frequency. By way of example only,modified nucleotides may have incorporation blockers that may bedeblocked by a combination of pH and light. For instance, where a flowcell 700 may control pH between two values and control light between twovalues, the flow cell 700 may selectively deblock the incorporationblocker of a given nucleotide, thereby making the nucleotide capable ofincorporation by an enzyme. One example of a type of incorporationblocker may include a chemical moiety that prevents the blocked modifiednucleotide from entering the active pocket of the enzyme. In someinstances, the modified nucleotide may also include an extensionblocker.

To achieve selective activation or deposition as described above, theflow cell 700 may selectively activate the light sources 722 and/or thepH control feature 740. For instance, if a particular nucleotide baserequires exposure to a certain pH value in combination with exposure tolight at a certain frequency, and if it desirable to add that nucleotidebase to a polynucleotide in a given well 730, the pH control feature 740and light source 722 of that well 730 may be driven to provide thatparticular combination of pH value and light frequency to therebyactivate/deposit that particular nucleotide base. To complete amachine-written polynucleotide in a given well 730, the flow cell 700may selectively drive the pH control feature 740 and light source 722for that well 730 to provide a particular sequence of pH values andlight frequencies in accordance with a desired corresponding sequence ofnucleotide bases to thereby attach those nucleotide bases to a primer atthe bottom of the well 730 in the desired sequence.

In the foregoing example, the flow cell 700 provides selectiveactivation/deposition of nucleotide bases based on a combination of pHvalues and light frequencies associated with particular nucleotidebases. In some other versions, the flow cell 700 provides selectiveactivation/deposition of nucleotide bases based solely on pH valuesassociated with particular nucleotide bases. In such versions, the flowcell 700 drives the pH control feature 740 for a given well 730 toprovide a particular sequence of pH values in accordance with a desiredcorresponding sequence of nucleotide bases to thereby attach thosenucleotide bases to a primer at the bottom of the well 730 in thedesired sequence. In such versions, the light source 722 may be usedsolely for a reading stage or may be omitted altogether.

As yet another illustrative variation, the flow cell 700 may provideselective activation/deposition of nucleotide bases based solely onlight frequencies associated with particular nucleotide bases. In suchversions, the flow cell 700 drives the light source 722 for a given well730 to provide a particular sequence of light frequencies in accordancewith a desired corresponding sequence of nucleotide bases to therebyattach those nucleotide bases to a primer at the bottom of the well 730in the desired sequence. In such versions, pH control feature 740 may beomitted altogether.

It should be understood from the foregoing that, in versions of the flowcell 700 that include light sources 722, such light sources 722 may beutilized both during a writing stage (e.g., when light frequencies aloneor in combination with pH values cause selective activation/depositionof nucleotides) and during a reading stage (e.g., similar to lightsource 560 described above in the context of flow cells 500, 601).Alternatively, two separate light sources may be used—one that is onlyused during a writing stage and another that is used only during areading stage. As yet another illustrative alternative, the flow cell700 may be a “write only” flow cell where the image sensors 792 areomitted such that the light sources 722 are only used during a writingprocess.

B. Flow Cell with Illumination Assembly for Activation of Nucleotides

As noted above, light (alone or in combination with pH values) may beutilized to selectively activate or deposit nucleotides. For instance,different nucleotides may be activated or deposited in response toexposure to different light frequencies. FIG. 12 shows another exampleof a flow cell 800 that may be utilized to provide machine-writing ofpolynucleotides by providing selectively activated sequences of lightfrequencies to thereby selectively activate corresponding nucleotidebases during a DNA machine-writing process.

The flow cell 800 of the example shown in FIG. 12 includes an upper bodyportion 802, a middle body portion 804, and a lower body portion 806. Anupper fluid flow channel 864 is defined between the upper and middlebody portions 802, 804 and is operable to receive a flow of fluid (e.g.,a fluid containing nucleotide bases, etc.). A lower fluid flow channel862 is defined between the middle and lower body portions 804, 806 andis operable to receive a separate flow of fluid (e.g., a fluidcontaining deblocking/deshielding agents, etc.). As with lower fluidflow channel 662 described above, lower fluid flow channel 862 of thisexample may be optional and may be omitted in some variations.

The flow cell 800 of this example further includes a plurality of wells830 that are formed as recesses in the bottom surface of the upper fluidflow channel 864. These wells 830 are at least substantially similar tothe wells 630 described above. An illumination assembly 840 ispositioned at the bottom 832 of each well 830. The illumination assembly840 includes a microLED panel 844 and a polarizer grid 842 laid over themicroLED panel 842. By way of example only, each microLED panel 844 mayinclude a plurality of microLEDs arranged in a grid or other pattern,with each microLED being operable to emit light at a particularfrequency. While the microLED panel 844 is located at the bottom 832 ofeach well 830 in this example, the microLED panel 844 may instead belocated elsewhere. For instance, the microLED panel 844 may instead belocated in or on the sidewall 834 of each well 830. As anotherillustrative alternative, each microLED panel 844 may be located on orin the upper surface 824 of the upper flow channel 824, directly abovethe corresponding well 830. In versions where the microLED panel 844 islocated in or on the sidewall 834, or on or in the upper surface 824,each polarizer grid 842 may be correspondingly located or may beomitted.

The polarizer grid 842 may include an array of polarizing elements thatmay be selectively activated to serve as shutters over correspondingmicroLEDs of the microLED panel 844. In other words, the number andarrangement of polarizing elements in the polarizer grid 842 maycorrespond directly with the number and arrangement of microLEDs in themicroLED panel 844. The polarizing grid 842 may be activated to permitlight emitted by a selected microLED of the microLED panel 844 to onlyreach a corresponding region of the surface area at the bottom of thewell 830. To achieve this, the polarizing grid 842 may effectively“open” only the “shutter” provided by the polarizing element that isdirectly over the activated microLED of the microLED panel 844, leavingthe rest of the “shutters” provided by the rest of the polarizingelements in a “closed” state. This may provide greater precision in thedelivery of light to the bottom surface of the well 830, preventing thelight from undesirably reaching regions of the bottom surface of thewell 830 that are adjacent to the targeted region. It should beunderstood that the polarizing grid 842 may be optional. Othervariations of the flow cell 800 may lack the polarizing grid 842.

In the present example, the bottom 832 of each well 830 also includes anopening 860 providing a pathway for fluid communication between the well830 and the lower fluid flow channel 862. In some versions, this opening860 includes a valve that is operable to selectively open or close tothereby selectively permit or prevent fluid communication from the lowerfluid flow channel 862 to the corresponding well 830. As noted above,the lower fluid flow channel 862 may be optional. Likewise, the opening860 (and corresponding valve, if any) may be optional. Some variationsof the flow cell 800 may lack the opening 860 at the bottom of each well830.

As described above, light may be utilized to machine-writepolynucleotides. In particular, nucleotides may be selectively activatedor deposited based on exposure to light having a certain frequency. Toachieve this activation/deposition, the flow cell 800 may selectivelyactivate the illumination assembly 840. For instance, if a particularnucleotide base requires exposure to light at a certain frequency, andif it desirable to add that nucleotide base to a polynucleotide in agiven well 830, the illumination assembly 840 of that well 830 may bedriven to provide that particular light frequency at a particular regionon the bottom 832 of the well 830 to thereby activate/deposit thatparticular nucleotide base at that particular region. To complete amachine-written polynucleotide in a given well 830, the flow cell 800may selectively drive the illumination assembly 840 for that well 830 toprovide a particular sequence of light frequencies in accordance with adesired corresponding sequence of nucleotide bases to thereby attachthose nucleotide bases to a primer at the corresponding region of thebottom 832 of the well 830 in the desired sequence.

While the flow cell 800 of this example lacks a pH control feature likethe pH control feature 740 of the flow cell 700 described above withreference to FIG. 11, other variations of the flow cell 800 of FIG. 12may include a pH control feature like the pH control feature 740 of theflow cell 700. Such a pH control feature may be operated in cooperationwith the illumination assembly 840 to selectively activate/depositnucleotide bases based on a combination of pH values and lightfrequencies associated with particular nucleotide bases.

As also described above, light may be utilized to read machine-writtenpolynucleotides within the wells 830. To that end, the same illuminationassemblies 840 that are used to provide machine-writing ofpolynucleotides may be used to provide reading of machine-writtenpolynucleotides. In other words, the microLED panel 844 may be activatedto illuminate machine-written polynucleotides in a corresponding well830, and fluorophores associated with those machine-writtenpolynucleotides may fluoresce in response to such light. Thefluorescence profile of the fluorophores associated with themachine-written polynucleotides may enable reading of the nucleotidesequences of those machine-written polynucleotides. To pick up suchfluorescence, the flow cell 800 of the present example further includesa lower integrated circuit layer 890 with a plurality of image sensors892. By way of example only, the lower integrated circuit layer 890 andimage sensors 892 may be part of a CMOS chip.

The lower integrated circuit layer 890 and image sensors 892 areintegrated into the lower body portion 806 in this example, with theimage sensors 892 being positioned at the lower surface 866 of the lowerfluid flow channel 762. In some other variations, the lower integratedcircuit layer 890 and image sensors 892 are integrated into some othercomponent. By way of example only, the lower integrated circuit layer890 and/or image sensors 892 may be integrated into a cartridge (e.g.,the removable cartridge 200) that receives the flow cell 800; may beintegrated into the base instrument 102; or may be integrated in someother component.

Each image sensor 892 is positioned under a corresponding well 830.Thus, when the microLED panel(s) 844 is/are activated to emit lighttoward the well(s) 830 during a reading stage, the corresponding imagesensor(s) 892 is/are configured to detect fluorescence emitted byfluorophores associated with polynucleotides (e.g., machine-written DNA)contained within the well(s) 830. The fluorescent light profile detectedby image sensors 892 may be utilized to read the polynucleotides asdescribed herein.

It should be understood from the foregoing that microLED panels 844 maybe utilized both during a writing stage (e.g., when light frequenciescause selective activation/deposition of nucleotides) and during areading stage (e.g., similar to light source 560 described above in thecontext of flow cells 500, 601). Alternatively, two separate lightsources may be used—one that is only used during a writing stage andanother that is used only during a reading stage. As yet anotherillustrative alternative, the flow cell 800 may be a “write only” flowcell where the image sensors 892 are omitted such that the microLEDpanels 844 are only used during a writing process.

C. Printhead for Selective Delivery of Nucleotide Bases

As another illustrative example, a printhead may be used to selectivelydeposit nucleotides or nucleobases (e.g., similar to an inkjet printeror additive manufacturing 3D printer, etc.) within a well of a flow cellto thereby machine-write polynucleotides. FIG. 13 shows an example ofhow this may be carried out. In particular, FIG. 13 shows a flow cell900 that includes an upper body portion 902 and a lower body portion904. An upper fluid flow channel 910 is defined between the upper andmiddle body portions 902, 904. The flow cell 900 of this example lacks alower fluid flow channel. In some other variations, the flow cell 900may include a lower fluid flow channel (e.g., similar to the lower fluidflow channel 662 described above).

The flow cell 900 of this example further includes a plurality of wells930 that are formed as recesses in the bottom surface of the upper fluidflow channel 910. While only one well 930 is shown in FIG. 13, it shouldbe understood that the flow cell 900 may include any suitable number ofwells 930 in any suitable arrangement. The bottom 932 of the well 930includes an electrode assembly 940 that is formed by four electrodes942, 944, 946, 948. An example of a manner in which the electrodeassembly 940 may be operated will be described in greater detail below.It should be understood, however, that electrode assembly 940 may beoptional; and that the electrode assembly 940 may be omitted in somevariations. It should also be understood that, in variations where theflow cell 900 includes a lower fluid flow channel (e.g., similar to thelower fluid flow channel 662 described above), each well 930 may furtherinclude an opening providing a pathway for fluid communication betweenthe well 930 and the lower fluid flow channel (e.g., with or without avalve in such an opening).

In the present example, a guide rail 920 is positioned above the lowerbody portion 904 of the flow cell 900. A printhead 950 is slidablydisposed along the guide rail 920. In some versions, the guide rail 920and the printhead 950 are integrated into the flow cell 900. In someother versions, the guide rail 920 and the printhead 950 are integratedinto a cartridge (e.g., the removable cartridge 200) that receives theflow cell 900. In still other versions, the guide rail 920 and theprinthead 950 are integrated into another piece of equipment (e.g., thebase instrument 102, etc.). While only one printhead 950 is shown in thepresent example, other versions of flow cell 900 may have more than oneprinthead 950 (e.g., an array of printheads 950). Some such versions mayhave one printhead 950 per well 930. In some such versions, eachprinthead 950 may remain stationary relative to the wells 930.

The printhead 950 is operable to travel along the guide rail 920 tothere by selectively position the printhead 950 over various wells 930within the flow cell 900. By way of example only, the printhead 950 mayinclude an integrated motor that drives a pinion, with the pinion beingengaged with a rack presented by the guide rail 920. Other suitable waysin which the printhead 950 may selectively travel along the guide rail920 will be apparent to those skilled in the art in view of theteachings herein. It should also be understood that the guide rail 920may be optional; and that various other kinds of components may be usedto provide guided movement of the printhead 950 in relation to the wells930 of the flow cell 900. Such variations may further enable provideguided movement of the printhead 950 along at last two axes in relationto the wells 930, such that the printhead 950 may be selectivelypositioned over wells 930 in different rows and columns in the flow cell900. Again, other suitable ways in which the printhead 950 mayselectively travel in relation to the wells 930 will be apparent tothose skilled in the art in view of the teachings herein.

The printhead 950 of the present example includes an integral controller952, a set of nucleobase reservoirs 960, 962, 964, 966, and a set ofnozzles 970, 972, 974, 976. The controller 952 may include an integratedcircuit and/or any other suitable components as will be apparent tothose skilled in the art in view of the teachings herein. Each nozzle970, 972, 974, 976 is associated with a corresponding one of thenucleobase reservoirs 960, 962, 964, 966. The controller 952 is operableto drive the printhead 950 to expel one particular nucleobase (e.g.,adenine) from the nucleobase reservoir 960 out through the nozzle 970;another particular nucleobase (e.g., cytosine) from the nucleobasereservoir 962 out through the nozzle 972; another particular nucleobase(e.g., guanine) from the nucleobase reservoir 964 out through the nozzle974; and another particular nucleobase (e.g., thymine) from thenucleobase reservoir 966 out through the nozzle 976. Various suitableways in which this may be accomplished will be apparent to those skilledin the art in view of the teachings herein. By way of example only, theprinthead 950 may selectively expel nucleobases through correspondingnozzles 970, 972, 974, 976 using components and techniques similar tothose employed in conventional inkjet printers or additive manufacturing3D printers, etc.

While each nucleobase is expelled through a respective nozzle 970, 972,974, 976 in this example, other variations may provide a manifoldbetween the nucleobase reservoirs 960, 962, 964, 966 and a single,shared nozzle. All four nucleobases may thus be expelled from theprinthead via the same nozzle. In such versions, the printhead mayfurther include a means for purging the manifold and corresponding fluidline to the shared nozzle, with such purging being provided when theprinthead is switching over from expelling one nucleobase through theshared nozzle to expelling another nucleobase through the shared nozzle.

It should also be understood that other variations of the printhead 950may lack the integrated nucleobase reservoirs 960, 962, 964, 966. Insome such variations, the nucleobase reservoirs 960, 962, 964, 966 arelocated elsewhere (e.g., in a cartridge that receives the flow cell 900,in the base instrument 102, etc.). In such variations, flexible fluidlines may couple the nucleobase reservoirs 960, 962, 964, 966 with thecorresponding nozzles 970, 972, 974, 976 (or with a manifold that leadsto a shared nozzle) of the printhead 950. Such flexible fluid lines maystill permit the printhead 950 to move freely relative to the wells 930.Moreover, such flexible fluid lines may still permit the printhead 950to move freely relative to the nucleobase reservoirs 960, 962, 964, 966when the nucleobase reservoirs 960, 962, 964, 966 are not integratedinto the printhead 950.

It should be understood from the foregoing, that the printhead 950 maybe selectively positioned over a particular well 930; then be activatedto selectively expel nucleobases from the nozzles 970, 972, 974, 976 ina particular sequence in order to machine-write a polynucleotide. Insome versions, the printhead 950 moves in relation to the well 930between each nucleobase expulsion, in order to successively positioneach nozzle 970, 972, 974, 976 along the same vertical axis when thatnozzle 970, 972, 974, 976 is activated to expel a nucleobase. This maypromote deposition of nucleotides in a strand that is aligned with theaxis at which each nozzle 970, 972, 974, 976 is activated.

As noted above, each well 930 of the present example further includes anelectrode assembly 940 that is formed by four electrodes 942, 944, 946,948. In this example, each electrode 942, 944, 946, 948 is associatedwith a particular nucleobase (e.g., similar to the electrode segments642, 644, 646, 648 described above in the context of FIG. 7). Forinstance, electrode 942 may be configured to provide a charge that isuniquely associated with adenine; electrode 944 may be configured toprovide a charge that is uniquely associated with cytosine; electrode946 may be configured to provide a charge that is uniquely associatedwith guanine; and electrode 948 may be configured to provide a chargethat is uniquely associated with thymine. Following the above examplewhere certain nucleobases are associated with certain correspondingnozzles 970, 972, 974, 976, the electrode 942 may be activated whenadenine is expelled through the nozzle 970; the electrode 944 may beactivated when cytosine is expelled through the nozzle 972; theelectrode 946 may be activated when guanine is expelled through thenozzle 974; and the electrode 948 may be activated when thymine isexpelled through the nozzle 976. Such activation of the electrodes 942,944, 946, 948 in cooperation with expulsion from the nozzles 970, 972,974, 976 may further promote formation of machine-writtenpolynucleotides with nucleobases in the desired sequence. In some otherversions, the electrode assembly 940 has only one electrode that isoperable to activate any nucleobase that is emitted by the printhead950.

FIG. 13 also shows an acoustic tamping assembly 980 movably positionedon the guide rail 920. The acoustic tamping assembly 980 may have thesame kinds of relationships with the guide rail 920 as described abovewith respect to the printhead 950. The acoustic tamping assembly 980 mayalso be configured to move in relation to the wells 930 in any of theother manners described above with respect to the printhead 950. Theacoustic tamping assembly 980 of this example includes an integralcontroller 982 and an acoustic transducer 984. The controller 982 mayinclude an integrated circuit and/or any other suitable components aswill be apparent to those skilled in the art in view of the teachingsherein. The controller 982 is operable to selectively drive the acoustictransducer 984 to emit acoustic waves downwardly toward the well 930.Various suitable forms that the acoustic transducer 984 may take will beapparent to those skilled in the art in view of the teachings herein. Itshould also be understood that the acoustic tamping assembly 980 may beintegrated into the printhead 950, such that it is not necessarilyrequired to provide the acoustic tamping assembly 980 and the printhead950 as separate components. Moreover, the acoustic tamping assembly 980may be omitted altogether in some versions.

In operation, the printhead 950 may first be activated deposit one ormore nucleobases in a well 930; and the acoustic tamping assembly 980may then be activated to acoustically tamp the deposited nucleobase(s)in the well 930. For instance, the printhead 950 may be positioned overa particular region of the well 930 to deposit the selectednucleobase(s); then the printhead 950 may be moved out of the way; thenthe acoustic tamping assembly 980 may be moved into place over thatparticular region of the well 930 to acoustically tamp the depositednucleobase. This acoustic tamping may ensure that the nucleobase emittedby the printhead 950 reaches the intended well 930. The acoustic tampingmay also prevent diffusion from occurring between the wells 930. Inversions where the acoustic tamping assembly 980 is integrated directlyinto the printhead 950, a sequence of movement of the printhead 950 andthen the acoustic tamping assembly 980 may not be necessary.

In addition to the foregoing, any of the flow cells and writingprocesses described herein may utilize a photoacid (e.g., changing thepH within a well by activating an acid with light). In addition, or inthe alternative, any of the flow cells and writing processes describedherein may utilize methylation to increase encoding space (e.g.,allowing use of methylated adenine, methylated cytosine, methylatedguanine, and/or methylated thymine in addition to of adenine, cytosine,guanine, and thymine). In addition, or in the alternative, any of theflow cells and writing processes described herein may utilize selectivedehybridization, where an enzyme is hybridized to inhibitor (e.g.,providing selective inhibition and/or anchoring an enzyme to a spatiallylocalized region). In addition, or in the alternative, any of the flowcells and writing processes described herein may utilize varyingcurrents to draw polynucleotides to a desired location or release thepolynucleotide (e.g., to assist in transferring polynucleotides from oneregion of space to another region of space; or from one device toanother device). In addition, or in the alternative, any of the flowcells and writing processes described herein may utilize pre-chargedenzymes to provide selective deposition or activation of particularnucleotides.

VIII. Miscellaneous

All of the references, including patents, patent applications, andarticles, are explicitly incorporated by reference herein in theirentirety.

The foregoing description is provided to enable a person skilled in theart to practice the various configurations described herein. While thesubject technology has been particularly described with reference to thevarious figures and configurations, it should be understood that theseare for illustration purposes only and should not be taken as limitingthe scope of the subject technology.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one implementation” are not intended to beinterpreted as excluding the existence of additional implementationsthat also incorporate the recited features. Moreover, unless explicitlystated to the contrary, implementations “comprising” or “having” anelement or a plurality of elements having a particular property mayinclude additional elements whether or not they have that property.

The terms “substantially” and “about” used throughout this Specificationare used to describe and account for small fluctuations, such as due tovariations in processing. For example, they may refer to less than orequal to ±5%, such as less than or equal to ±2%, such as less than orequal to ±1%, such as less than or equal to ±0.5%, such as less than orequal to ±0.2%, such as less than or equal to ±0.1%, such as less thanor equal to ±0.05%.

There may be many other ways to implement the subject technology.Various functions and elements described herein may be partitioneddifferently from those shown without departing from the scope of thesubject technology. Various modifications to these implementations maybe readily apparent to those skilled in the art, and generic principlesdefined herein may be applied to other implementations. Thus, manychanges and modifications may be made to the subject technology, by onehaving ordinary skill in the art, without departing from the scope ofthe subject technology. For instance, different numbers of a givenmodule or unit may be employed, a different type or types of a givenmodule or unit may be employed, a given module or unit may be added, ora given module or unit may be omitted.

Underlined and/or italicized headings and subheadings are used forconvenience only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. All structural and functional equivalents to theelements of the various implementations described throughout thisdisclosure that are known or later come to be known to those of ordinaryskill in the art are expressly incorporated herein by reference andintended to be encompassed by the subject technology. Moreover, nothingdisclosed herein is intended to be dedicated to the public regardless ofwhether such disclosure is explicitly recited in the above description.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

What is claimed is:
 1. An apparatus, comprising: (a) a flow cell body defining one or more flow channels and a plurality of wells, each flow channel of the one or more flow channels to receive a flow of fluid, each well of the plurality of wells being fluidically coupled with the corresponding flow channel of the one or more flow channels, each well of the plurality of wells to contain at least one polynucleotide; (b) a plurality of electrodes, each electrode of the plurality of electrodes being positioned in a corresponding well of the plurality of wells, the plurality of electrodes to effect writing of polynucleotides in the corresponding wells of the plurality of wells; (c) an integrated circuit, the integrated circuit to drive selective deposition or activation of selected nucleotides to attach to polynucleotides in the wells of the plurality of wells to thereby generate polynucleotides representing machine-written data in the plurality of wells; and (d) an imaging assembly to capture images indicative of one or more nucleotides in a polynucleotide.
 2. The apparatus of claim 1, each electrode of the plurality of electrodes defining an aperture.
 3. The apparatus of claim 2, the imaging assembly including at least one image sensor to receive light through the aperture of each electrode of the plurality of electrodes.
 4. The apparatus of any one or more of claims 2 through 3, each electrode of the plurality of electrodes being annularly shaped.
 5. The apparatus of any one or more of claims 2 through 3, each electrode of the plurality of electrodes comprising a plurality of electrode segments arranged in quadrants, the aperture being defined at a central region of the arrangement of quadrants.
 6. The apparatus of claim 5, the integrated circuit being further in communication with the imaging assembly.
 7. The apparatus of any one or more of claims 1 through 6, the integrated circuit comprising a CMOS chip.
 8. The apparatus of any one or more of claims 1 through 7, the plurality of wells being formed as a plurality of discrete recesses arranged in a pattern along a floor of the corresponding flow channel of the one or more flow channels.
 9. The apparatus of any one or more of claims 1 through 8, each well of the plurality of wells being defined by at least one sidewall and a floor.
 10. The apparatus of claim 9, each electrode of the plurality of electrodes being positioned on the floor of the corresponding well of the plurality of wells.
 11. The apparatus of claim 9, each electrode of the plurality of electrodes being positioned on a sidewall of the at least one sidewall of the corresponding well.
 12. The apparatus of any one or more of claims 9 through 11, the floor of each well of the plurality of wells further defining an aperture, the aperture to provide a path for fluid communication between the well of the plurality of wells and a fluid source.
 13. The apparatus of any one or more of claims 1 through 12, the integrated circuit to drive the plurality of electrodes to selectively deposit or activate selected nucleotides by applying a voltage within the corresponding well of the plurality of wells.
 14. The apparatus of claim 13, each nucleotide being associated with a particular voltage, the integrated circuit to drive the electrodes of the plurality of electrodes to selectively deposit or activate a selected nucleotide by applying the particular voltage associated with the selected nucleotide.
 15. The apparatus of claim 14, each well of the plurality of wells including a set of four electrodes from the plurality of electrodes, each electrode in the set of four being associated with a corresponding voltage of the particular voltages associated with the nucleotides, such that each electrode in the set of four corresponds with a particular one of four nucleotides.
 16. The apparatus of any one or more of claims 1 through 15, the integrated circuit to drive the selective deposition or activation of selected nucleotides by applying a change in pH within the corresponding well of the plurality of wells.
 17. The apparatus of any one or more of claims 1 through 16, further comprising at least one light source, the integrated circuit to drive the selective deposition or activation of selected nucleotides by activating the at least one light source.
 18. The apparatus of claim 17, each nucleotide being associated with a particular wavelength of light, the integrated circuit to drive the at least one light source to selectively deposit or activate a selected nucleotide by applying the particular wavelength of light associated with the selected nucleotide.
 19. The apparatus of any one or more of claims 17 through 18, the integrated circuit to drive the selective deposition or activation of selected nucleotides by applying a change in pH within the corresponding well of the plurality of wells in addition to driving the activation of the at least one light source.
 20. The apparatus of any one or more of claims 17 through 18, the at least one light source comprising a light matrix.
 21. The apparatus of claim 20, the light matrix comprising a matrix of microscopic light emitting diodes.
 22. The apparatus of any one or more of claims 20 through 21, the light matrix to project light onto a bottom of each well of the plurality of wells.
 23. The apparatus of any one or more of claims 20 through 22, the light matrix being positioned under a bottom of each well of the plurality of wells.
 24. The apparatus of any one or more of claims 17 through 23, further comprising one or more polarizers, the integrated circuit to drive the selective deposition or activation of selected nucleotides by activating the at least one polarizer of the one or more polarizers in coordination with the at least one light source.
 25. The apparatus of any one or more of claims 1 through 24, the integrated circuit to drive the selective deposition or activation of selected nucleotides by activating communication of pre-charged enzymes to the one or more flow channels.
 26. The apparatus of any one or more of claims 1 through 24, further comprising a printhead, the integrated circuit to drive the selective deposition or activation of selected nucleotides by activating the printhead.
 27. The apparatus of claim 26, the printhead including four nozzles, each nozzle of the four nozzles to dispense a corresponding nucleotide.
 28. The apparatus of any one or more of claims 26 through 27, the integrated circuit further to drive acoustic tamping of droplets emitted by the printhead.
 29. The apparatus of any one or more of claims 26 through 27, the integrated circuit further to activate the printhead and the plurality of electrodes in cooperation to thereby drive the selective deposition or activation of selected nucleotides.
 30. An apparatus, comprising: (a) a flow cell body defining one or more flow channels and a plurality of wells, each flow channel of the one or more flow channels to receive a flow of fluid, each well of the plurality of wells being fluidically coupled with the corresponding flow channel of the one or more flow channels, each well of the plurality of wells to contain at least one polynucleotide; (b) a plurality of electrodes, each electrode of the plurality of electrodes being positioned in a corresponding well of the plurality of wells, the plurality of electrodes to effect writing of polynucleotides in the corresponding wells of the plurality of wells; and (c) an integrated circuit, the integrated circuit to drive selective deposition or activation of selected nucleotides to attach to polynucleotides in the plurality of wells to thereby generate polynucleotides representing machine-written data in the plurality of wells, each nucleotide being associated with a particular voltage, the integrated circuit to drive the plurality of electrodes to selectively deposit or activate a selected nucleotide by applying the particular voltage associated with the selected nucleotide.
 31. The apparatus of claim 30, each well of the plurality of wells including a set of four electrodes from the plurality of electrodes, each electrode in the set of four being associated with a corresponding voltage of the particular voltages associated with the nucleotides, such that each electrode in the set of four corresponds with a particular one of four nucleotides.
 32. An apparatus, comprising: (a) a flow cell body defining one or more flow channels and a plurality of wells, each flow channel of the one or more flow channels to receive a flow of fluid, each well of the plurality of wells being fluidically coupled with the corresponding flow channel of the one or more flow channels, each well of the plurality of wells to contain at least one polynucleotide; (b) a plurality of electrodes, each electrode of the plurality of electrodes being positioned in a corresponding well of the plurality of wells, the plurality of electrodes to effect writing of polynucleotides in the corresponding wells of the plurality of wells; (c) at least one light source; and (d) an integrated circuit, the integrated circuit to drive selective deposition or activation of selected nucleotides to attach to polynucleotides in the plurality of wells to thereby generate polynucleotides representing machine-written data in the plurality of wells, the integrated circuit to drive the selective deposition or activation of selected nucleotides by activating the at least one light source.
 33. The apparatus of claim 32, each nucleotide being associated with a particular wavelength of light, the integrated circuit to drive the at least one light source to selectively deposit or activate a selected nucleotide by applying the particular wavelength of light associated with the selected nucleotide.
 34. The apparatus of any one or more of claims 32 through 33, the integrated circuit to drive the selective deposition or activation of selected nucleotides by applying a change in pH within the corresponding well of the plurality of wells in addition to driving the activation of the at least one light source.
 35. The apparatus of any one or more of claims 32 through 34, the at least one light source comprising a light matrix.
 36. The apparatus of claim 35, the light matrix comprising a matrix of micro-LEDs.
 37. The apparatus of any one or more of claims 35 through 36, the light matrix to project light onto a bottom of each well of the plurality of wells.
 38. The apparatus of any one or more of claims 35 through 37, the light matrix being positioned under a bottom of each well of the plurality of wells.
 39. The apparatus of any one or more of claims 32 through 38, further comprising one or more polarizers, the integrated circuit to drive the selective deposition or activation of selected nucleotides by activating the one or more polarizers in coordination with the at least one light source.
 40. An apparatus, comprising: (a) a flow cell body defining one or more flow channels and a plurality of wells, each flow channel of the one or more flow channels to receive a flow of fluid, each well of the plurality of wells being fluidically coupled with the corresponding flow channel of the one or more flow channels, each well of the plurality of wells to contain at least one polynucleotide; (b) a plurality of electrodes, each electrode of the plurality of electrodes being positioned in a corresponding well of the plurality of wells, the plurality of electrodes to effect writing of polynucleotides in the corresponding wells of the plurality of wells; (c) a printhead to deposit nucleotides; and (d) an integrated circuit, the integrated circuit to drive selective deposition of selected nucleotides to attach to polynucleotides in the plurality of wells to thereby generate polynucleotides representing machine-written data in the plurality of wells, the integrated circuit to drive the selective deposition of selected nucleotides by activating the printhead.
 41. The apparatus of claim 40, the printhead including four nozzles, each nozzle to dispense a corresponding nucleotide.
 42. The apparatus of any one or more of claims 40 through 41, the integrated circuit further to drive acoustic tamping of droplets emitted by the printhead.
 43. The apparatus of any one or more of claims 40 through 42, the integrated circuit further to activate the printhead and the plurality of electrodes in cooperation to thereby drive the selective deposition or activation of selected nucleotides.
 44. A method comprising: flowing a fluid through a flow channel of a flow cell, the fluid comprising a plurality of types of nucleotides, the flow cell including a plurality of primary bases to support polynucleotides, the primary bases being secured to a floor region of the flow cell; selecting one type of nucleotide for attachment to a selected primary base of the plurality of primary bases in the flow cell; and activating the selected one type of nucleotide to thereby cause the selected one type of nucleotide to attach to the selected primary base, the attached nucleotide representing machine-written data.
 45. The method of claim 44, further comprising repeating selecting one type of nucleotide for attachment to a selected primary base in the flow cell and activating the selected one type of nucleotide to thereby cause the selected one type of nucleotide to attach to the selected primary base, to thereby generate a polynucleotide on the primary base, the generated polynucleotide including a plurality of selected types of nucleotides, the generated polynucleotide representing machine-written data.
 46. The method of any one or more of claims 44 through 45, activating the selected type of nucleotide comprising activating an electrode in the flow cell to thereby apply a voltage to the selected type of nucleotide.
 47. The method of claim 46, each type of nucleotide being associated with a particular voltage, activating the electrode comprising activating the electrode to apply the particular voltage associated with the selected type of nucleotide.
 48. The method of claim 47, the flow cell including electrodes associated with different corresponding voltages, the method further comprising selecting an electrode from the electrodes, the selected electrode corresponding to the voltage associated with the selected type of nucleotide, the activated electrode being the selected electrode.
 49. The method of any one or more of claims 44 through 48, activating the selected type of nucleotide comprising applying a change in pH within the flow cell, the applied pH being associated with the selected type of nucleotide.
 50. The method of any one or more of claims 44 through 48, activating the selected type of nucleotide comprising activating at least one light source.
 51. The method of claim 50, each type of nucleotide being associated with a particular wavelength of light, activating at least one light source comprising activating the at least one light source to emit light at the particular wavelength associated with the selected type of nucleotide.
 52. The method of any one or more of claims 50 through 51, activating the selected type of nucleotide further comprising applying a change in pH within the flow cell in coordination with activating the at least one light source, the applied pH being associated with the selected type of nucleotide.
 53. The method of any one or more of claims 50 through 52, activating the selected type of nucleotide further comprising activating at least one polarizer in coordination with activating the at least one light source.
 54. The method of any one or more of claims 44 through 53, activating the selected type of nucleotide comprising communicating pre-charged enzymes to the flow channel.
 55. A method comprising: selecting one type of nucleotide for attachment to a selected primary base in a flow cell; and depositing the selected one type of nucleotide into the flow cell, the flow cell including a plurality of primary bases to support polynucleotides, the primary bases being secured to a floor region of the flow cell, the deposited nucleotide attaching to a corresponding primary base of the plurality of primary bases, the attached nucleotide representing machine-written data.
 56. The method of claim 55, depositing the selected type of nucleotide comprising emitting the selected type of nucleotide from a printhead.
 57. The method of claim 56, the printhead including a plurality of nozzles, each nozzle of the plurality of nozzles being associated with a particular type of nucleotide, the method further comprising selecting the nozzle of the plurality of nozzles corresponding to the selected type of nucleotide, the deposited nucleotide being deposited from the selected nozzle of the plurality of nozzles.
 58. The method of any one or more of claims 56 through 57, depositing the selected type of nucleotide further comprising activating an electrode in the flow cell in coordination with emitting the selected type of nucleotide from the printhead.
 59. The method of claim 58, the flow cell including electrodes associated with different corresponding voltages, each type of nucleotide being associated with a particular voltage, the method further comprising selecting an electrode of the electrodes, the selected electrode corresponding to the voltage associated with the selected type of nucleotide, the activated electrode being the selected electrode. 